Methods and compositions for treatment of human immunodeficiency virus infection with conjugated antibodies or antibody fragments

ABSTRACT

The present invention concerns methods and compositions for treatment of HIV infection in a subject. The compositions may comprise a targeting molecule against an HIV antigen, such as an anti-HIV antibody or antibody fragment. The anti-HIV antibody or fragment may be conjugated to a variety of cytotoxic agents, such as doxorubicin. In a preferred embodiment, the antibody or fragment is P4/D10. Other embodiments may concern methods of imaging, detection or diagnosis of HIV infection in a subject using an anti-HIV antibody or fragment conjugated to a diagnostic agent. In alternative embodiments, a bispecific antibody with at least one binding site for an HIV antigen and at least one binding site for a carrier molecule may be administered, optionally followed by a clearing agent, followed by administration of a carrier molecule conjugated to a therapeutic agent.

RELATED APPLICATIONS

This is a divisional of U.S. application Ser. No. 11/745,692 (now issuedU.S. Pat. No. 8,333,971), filed May 8, 2007, which claims the benefitunder 35 C.F.R. §119(e) to provisional application Ser. No. 60/800,342,filed May 15, 2006, the entire contents of which are incorporated hereinby reference.

BACKGROUND

1. Field of the Invention

The present invention concerns methods and compositions for treating andin preferred embodiments eliminating human immunodeficiency virus (HIV)in infected subjects. In particular embodiments, the compositions andmethods concern targeting molecules, such as antibodies or antibodyfragments against HIV antigens, for example against HIV envelopeantigen. In more particular embodiments, the antibodies or antibodyfragments may be conjugated to one or more agents, such as therapeuticagents, diagnostic agents, virostatic agents and/or cytotoxic agents,including but not limited to chemotherapeutic agents such asdoxorubicin. In alternative embodiments, bispecific or multispecificantibodies or fragments thereof may be used, with one or more bindingsites directed towards HIV antigen(s) and one or more binding sites withaffinity for a carrier molecule to which cytotoxic, virostatic or othertherapeutic and/or diagnostic agents may be attached.

2. Description of Related Art

Despite encouraging advances in the treatment of human immunodeficiencyvirus-1 (HIV-1) with anti-retroviral therapy (ART), analyses ofperipheral blood and lymph nodes have documented the presence ofpersistent reservoirs of resting T cells which harbor latent provirusthat can activate spontaneously even years after the termination oftherapy (Berger et al., Proc Natl Acad Sci USA 1998, 95:11511-11513;Blankson et al., Annu Rev Med 2002, 53:557-593).

Binding and neutralizing antibodies can prevent attachment of free virusto the cellular receptor, they can bind to the viral surface, and theycan induce complement-mediated virolysis of free virions (Parren et al.,AIDS 1999, 13[Suppl A]:S137-162). Antibodies may also mediate killing ofinfected cells by antibody-dependent cellular cytotoxicity (ADCC), bycoupling NK-cells to infected target cells (Broliden et al., J Virol1990, 64:936-940). However, the use of anti-viral antibodies alone aspart of an immunotherapy of patients infected with HIV has not fulfilledits initial promise (Hinkula et al., J Acquir Immune Defic Syndr 1994,7:940-951; Trkola et al., Nat Med 2005, 11:615-622).

Attempts have been made to use various viral or cellular components astargets for antibody delivery of therapeutic agents to HIV-infectedcells (Davey et al., J Infect Dis 1994, 170:1180-1188; Pincus et al., JImmunol 2003, 170:2236-2241; Ramachandran et al., J Infect Dis 1994,170:1009-1013; Saavedra-Lozano et al., Proc Natl Acad Sci USA 2004,101:2494-2499). Similar immunotoxins have proved promising in cancerpatients (Wu and Senter, Nat Biotechnol 2005, 23:1137-1146). However, aneed exists for more effective methods and compositions for treatment ofHIV-infected cells.

SUMMARY OF THE INVENTION

The present invention fulfills an unresolved need in the art byproviding methods and compositions for inhibiting, suppressing,detecting, identifying, localizing and/or eliminating HIV-infectedcells. In certain embodiments, the compositions and/or methods mayconcern targeting molecules against HIV antigens. Such targetingmolecules may include, but are not limited to, peptides, antibodies,humanized antibodies, chimeric antibodies, human antibodies or fragmentsof any such antibodies, and/or antibody analogs. In certain embodiments,the targeting molecules may be unconjugated, for example “naked”antibodies or antibody fragments. In other embodiments, the targetingmolecules may be conjugated to one or more therapeutic and/or diagnosticagents. Such agents may include, but are not limited to, a drug,prodrug, virostatic agent, toxin, enzyme, oligonucleotide, radioisotope,radionuclide, immunomodulator, cytokine, label, fluorescent label,luminescent label, paramagnetic label, MRI label, micelle, liposome,nanoparticle, or combination thereof. In particular embodiments,conjugated anti-HIV antibodies or fragments may be administered in vivoto patients with a known or suspected HIV infection. Such administrationmay block or prevent infection of patient cells with HIV, may reduce oreliminate HIV-infected cells in the patient, and/or may reduce oreliminate residual foci of HIV-infected cells in patients treatedpreviously and/or simultaneously with other known anti-retroviraltherapies.

Other embodiments concern methods and/or compositions for treatingsubjects, such as subjects infected with HIV, SW, other retroviruses.Subjects may include, but are not limited to, humans, animals, cats,dogs, cows, sheep, goats, horses, and mammals. The methods andcompositions may comprise one or more naked or conjugated targetingmolecules to be administered to a subject. In preferred embodiments, thetargeting molecules are antibodies or antibody fragments, including anyvariation of chimeric, humanized or human antibodies or fragments.Administration may be by any route known in the art, such as oral,nasal, buccal, inhalational, rectal, vaginal or topical. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal, intraarterial, intrathecal orintravenous injection.

The skilled artisan will realize that one or more HIV targetingmolecules, either conjugated or unconjugated, may be administered aloneor alternatively in conjunction with other known therapeutic treatmentsfor HIV infection, such as azidothymidine, other nucleoside/nucleotidereverse transcriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, HIV protease inhibitors and/or fusion inhibitors. In certainembodiments, the conjugated HIV targeting molecules may be used incombination with HAART (highly active anti-retroviral therapy). Manyanti-HIV therapeutic agents are known in the art and any such knownagent may be used, including but not limited to efavirenz, zidovudine,tenofovir, lamivudine, emtricitabine, didanosine, abacavir, stavudine,nevirapine, lopinavir, ritonavir, atazanavir, fosamprenavir, indinavir,nelfinavir, saquinavir, alone or in any combination.

In some embodiments, anti-HIV antibodies or fragments may beadministered as part of a bispecific or multispecific antibody complex,with at least one binding site for the HIV antigen and a second bindingsite for a second target, such as a hapten or carrier molecule. In otherembodiments, anti-HIV antibodies or fragments may be covalently attachedto or provided as a fusion protein with an antibody, antibody fragment,monoclonal antibody, Fc fragment, Fc-binding protein or antibody bindingprotein.

In various embodiments, anti-HIV antibodies or fragments may becovalently or non-covalently attached to various moieties by methodswell known in the art, such as the use of covalent cross-linkingreagents. Many such agents, such as carbodiimides, bisimidates,N-hydroxysuccinimide ester of suberic acid,dimethyl-3,3′-dithio-bispropionimidate, azidoglyoxal,1,5-difluoro-2,4-(dinitrobenzene) and other cross-linkers of use forproteins and/or peptides are known and may be used.

In other embodiments, the anti-HIV antibodies or fragments may be usedas adjuncts for diagnosis and/or imaging purposes. For example, anti-HIVantibodies or fragments may be tagged with any known contrast ordetection agent or may be detected using any known methodologies, suchas ELISA, etc. The anti-HIV antibodies or fragments may be used ex vivo,for example by immunohistochemistry of tissue sections, to detectresidual HIV infection. Alternatively, anti-HIV antibodies or fragmentsmay be administered to a subject for in vivo detection of tissuesinfected with HIV. Such compositions and methods may be used to detectand/or diagnose the presence of HIV infection, to monitor for residualHIV infection after therapy, and/or to monitor the effectiveness ofanti-HIV therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of particularembodiments of the invention. The embodiments may be better understoodby reference to one or more of these drawings in combination with thedetailed description presented herein.

FIG. 1A. HIV-1_(IIIB) neutralization of HIV infection in vitro. Theneutralizing capacities of the immunoglobulins were tested by incubatingdifferent concentrations of the immunoglobulins with HIV-1_(IIIB) andthen assaying the viral infection of HIV susceptible Jurkat T-cells.Both 10 μg/ml doxorubicin-P4/D10 and unlabelled P4/D10 neutralizedHIV-1_(IIIB) significantly better than HIV negative sera (p=0.001).

FIG. 1B. HIV-1_(IIIB) inhibition of intercellular spread of HIVinfection in vitro. To test whether the immunoglobulins could limit theintercellular spread of HIV-1 infection Jurkat T-cells were mixed in theproportions 0.2%, 1%, 3%, and 5% infected and 99.8%, 99%, 97%, and 95%uninfected cells. The HIV-1 p24 production after treating 3% JurkatT-cells infected with HIV-1_(IIIB) and 97% uninfected cells withdifferent concentrations of immunoglobulins is shown. The results areshown as percent inhibition of p24 production after 7 days in culture.Doxorubicin-P4/D10 had a significantly better inhibiting effect onproduction of HIV-1 p24 compared to unlabelled P4/D10, control antibodydoxorubicin-LL1, free doxorubicin and HIV-negative serum at aconcentration of 0.5 or 0.05 μg/ml (p=0.002).

FIG. 2. Protection against HIV-1/MuLV infection in vivo. Mice(6-12/group) were challenged i.p. with HIV-1/MuLV infected splenocytesand immediately treated with monoclonal antibodies (Mab) or freedoxorubicin. Unconjugated P4/D10 Mab was titrated 100-800 μg per mouse,free doxorubicin 100-400 μg and irrelevant doxorubicin-hRS7 100-200 μg.All other treatments were given at 100 μg per mouse. Ten days afterchallenge, peritoneal cells were collected and mixed with HIVsusceptible Jurkat T-cells. HIV p24 production in these cell cultureswas measured every 3-4 days for 18 days. Percent of mice with a p24positive cell culture after treatment with 100 μg of Mab or freedoxorubicin is shown. Only cells from mice treated with 100 μgdoxorubicin-P4/D10 contained no infectious HIV, which was significantlydifferent (p=0.0001) from all other groups.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety.

DEFINITIONS

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, the terms “and” and “or” may be used to mean either theconjunctive or disjunctive. That is, both terms should be understood asequivalent to “and/or” unless otherwise stated.

As used herein, “about” means within plus or minus ten percent of anumber. For example, “about 100” would be mean any number between 90 and110.

An “antibody”, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion or analog of an immunoglobulin molecule, like an antibodyfragment.

An “antibody fragment” is a portion of an antibody such as F(ab)₂,F(ab′)₂, Fab, Fv, sFv, and the like. Regardless of structure, anantibody fragment binds with the same antigen that is recognized by theintact antibody. The term “antibody fragment” also includes anysynthetic or genetically engineered protein that acts like an antibodyby binding to a specific antigen to form a complex. For example,antibody fragments include isolated fragments consisting of the variableregions, such as the “Fv” fragments consisting of the variable regionsof the heavy and light chains, recombinant single chain polypeptidemolecules in which light and heavy variable regions are connected by apeptide linker (“scFv proteins”), and minimal recognition (CDR) unitsconsisting of the amino acid residues that mimic the hypervariableregion.

A “therapeutic agent” is an atom, molecule, or compound that is usefulin the treatment of a disease. Examples of therapeutic agents includeantibodies, antibody fragments, drugs, virostatic agents, toxins,enzymes, nucleases, hormones, immunomodulators, antisenseoligonucleotides, small interfering RNA (siRNA), chelators, boroncompounds, photoactive agents, dyes, and radioisotopes. Other exemplarytherapeutic agents and methods of use are disclosed in U.S. PatentApplication Publication Nos. 20050002945, 20040018557, 20030148409 and20050014207, each incorporated herein by reference.

A “neutralizing antibody” or “neutralizing antibody fragment” is usedherein to refer to an antibody or fragment that reacts with aninfectious agent (such as a virus) and destroys or inhibits itsinfectivity and/or virulence.

A “diagnostic agent” is an atom, molecule, or compound that is useful indiagnosing a disease. Useful diagnostic agents include, but are notlimited to, radioisotopes, dyes (such as with the biotin-streptavidincomplex), contrast agents, fluorescent compounds or molecules, andenhancing agents (e.g., paramagnetic ions) for magnetic resonanceimaging (MRI).

An “immunoconjugate” is a conjugate of a binding molecule (e.g., anantibody component) with an atom, molecule, or a higher-orderedstructure (e.g., with a carrier, a therapeutic agent, or a diagnosticagent).

A “naked antibody” is an antibody that is not conjugated to any otheragent.

A “carrier” is an atom, molecule, or higher-ordered structure that iscapable of associating with a therapeutic or diagnostic agent tofacilitate delivery of such agent to a targeted cell. Carriers mayinclude lipids (e.g., amphiphilic lipids that are capable of forminghigher-ordered structures), polysaccharides (such as dextran), proteins,peptides, peptide analogs, peptide derivatives or other higher-orderedstructures, such as micelles, liposomes, or nanoparticles. In certainembodiments, a carrier may be designed to be resistant to proteolytic orother enzymatic degradation, for example by substituting D-amino acidsfor naturally occurring L-amino acids in a protein or peptide.

As used herein, the term “antibody fusion protein” refers to arecombinantly produced antigen-binding molecule in which two or more ofthe same or different scFv or antibody fragments with the same ordifferent specificities are linked. Valency of the fusion proteinindicates how many binding arms or sites the fusion protein has to asingle antigen or epitope; i.e., monovalent, bivalent, trivalent ormultivalent. The multivalency of the antibody fusion protein means thatit can take advantage of multiple interactions in binding to an antigen,thus increasing the avidity of binding to the antigen. Specificityindicates how many antigens or epitopes an antibody fusion protein isable to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Monospecific, multivalent fusionproteins have more than one binding site for an epitope but only bindsto one such epitope, for example a diabody with two binding sitereactive with the same antigen. The fusion protein may comprise a singleantibody component, a multivalent or multispecific combination ofdifferent antibody components, or multiple copies of the same antibodycomponent. The fusion protein may additionally comprise an antibody oran antibody fragment and a therapeutic agent. Examples of therapeuticagents suitable for such fusion proteins include immunomodulators(“antibody-immunomodulator fusion protein”) and toxins (“antibody-toxinfusion protein”). One preferred toxin comprises a ribonuclease (RNase),preferably a recombinant RNase.

A “bispecific antibody” is an antibody that can bind simultaneously totwo targets of different structure. Bispecific antibodies and bispecificantibody fragments that are of particular interest have at least one armthat specifically binds to, for example, an HIV envelope protein and atleast one other arm that specifically binds to a targetable conjugatethat bears a therapeutic or diagnostic agent.

An antibody or immunoconjugate preparation, or a composition describedherein, is said to be administered in a “therapeutically effectiveamount” if the amount administered is physiologically significant. Anagent is physiologically significant if its presence results in adetectable change in the physiology of a recipient mammal. Inparticular, an anti-HIV antibody preparation is physiologicallysignificant if its presence reduces, inhibits or eliminates HIV-infectedcells or reduces, inhibits or eliminates HIV infection of non-infectedcells.

A composition is said to be a “pharmaceutically acceptable carrier” ifits administration can be tolerated by a recipient patient. Sterilephosphate-buffered saline is one example of a pharmaceuticallyacceptable carrier. Other suitable carriers are well known to those inthe art. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed.(Mack Publishing Co. 1995), and Goodman and Gilman's THE PHARMACOLOGICALBASIS OF THERAPEUTICS (Goodman et al., Eds. Macmillan Publishing Co.,New York, 1980 and 2001 editions).

Abbreviations used are:

ABS, sodium acetate buffer containing 150 mM sodium chloride;

ADCC, antibody-dependent cellular cytotoxicity;

DTT, dithiothreitol;

ELISA, enzyme-linked immunosorbent assay;

ART, anti-retroviral therapy;

HIV, human immunodeficiency virus;

Mab, monoclonal antibody;

MuLV, Murine Leukemia Virus;

PBMC, peripheral blood mononuclear cells;

TCID₅₀, 50% tissue culture infectious dose.

Antibodies

Various embodiments may concern antibody ligands against one or moreantigens or epitopes of HIV. In preferred embodiments, the antigen orepitope is one that is exposed on the surface of HIV-infected cells,such as the HIV envelope protein. Techniques for preparing and usingvarious antibody-based constructs and fragments are well known in theart. Means for preparing and characterizing antibodies are also wellknown in the art (See, e.g., Harlowe and Lane, 1988, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory). Antibodies of use mayalso be commercially obtained from a wide variety of known sources. Forexample, a variety of antibody secreting hybridoma lines are availablefrom the American Type Culture Collection (ATCC, Manassas, Va.).

Monoclonal Antibodies

While preferred embodiments may concern the use of the P4/D10 antibody,other anti-HIV antibodies may be obtained, prepared and/or used. Avariety of antibodies against HIV have been reported and in certainembodiments any such known anti-HIV antibody may be utilized. Forexample, 4E10 (Rosa et al., Immunity 2:163-73, 2005); 2F5 (Bryson etal., Protein and Peptide Letters, 8:413-18, 2001); 3D6 (Ruker et al.,Ann. NY Acad. Sci. 646:212-19, 1991); C37 (Cao et al., DNA and CellBiology, 12:836-41, 2004); LACY, 1F58, 1GGGC (Berry et al., Proteins,45:281-82, 2001); 2G12 (Armbruster et al., J. Antimicrob. Chemother.54:915-20, 2004), each incorporated herein by reference. In alternativeembodiments, monoclonal antibodies may be readily prepared through useof well-known techniques, such as those exemplified in U.S. Pat. No.4,196,265. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition. Cells from rodents such asmice and rats are preferred. Mice are more preferred, with the BALB/cmouse being most preferred as this is most routinely used and generallygives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B-lymphocytes (B-cells), are selected for usein the Mab generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Often, a panel of animals will have been immunized and thespleen of the animal with the highest antibody titer will be removed andthe spleen lymphocytes obtained by homogenizing the spleen with asyringe. Typically, a spleen from an immunized mouse containsapproximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B-lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art. For example, where the immunized animal is a mouse,one may use P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 41, Sp210-Ag14, FO,NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one mayuse R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2,LICR-LON-HMy2 and UC729-6 are all useful in connection with cellfusions.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus, and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, have been described. The use ofelectrically induced fusion methods is also appropriate.

Fusion procedures usually produce viable hybrids at low frequencies,around 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryagents are aminopterin, methotrexate, and azaserine. Aminopterin andmethotrexate block de novo synthesis of both purines and pyrimidines,whereas azaserine blocks only purine synthesis. Where aminopterin ormethotrexate is used, the media is supplemented with hypoxanthine andthymidine as a source of nucleotides (HAT medium). Where azaserine isused, the media is supplemented with hypoxanthine.

A preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two wk. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three wk) for the desired reactivity. The assay should be sensitive,simple and rapid, such as radioimmunoassays, enzyme immunoassays,cytotoxicity assays, plaque assays, dot immunobinding assays, and thelike.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide Mabs. The cell lines may be exploitedfor Mab production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide Mabs in high concentration. The individualcell lines also could be cultured in vitro, where the Mabs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. Mabs produced by either means may be furtherpurified, if desired, using filtration, centrifugation, and variouschromatographic methods such as HPLC or affinity chromatography.

Production of Antibody Fragments

Some embodiments of the claimed methods and/or compositions may concernantibody fragments. Such antibody fragments may be obtained by pepsin orpapain digestion of whole antibodies by conventional methods. Forexample, antibody fragments may be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)₂. Thisfragment may be further cleaved using a thiol reducing agent and,optionally, a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5S Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment. Exemplary methods forproducing antibody fragments are disclosed in U.S. Pat. No. 4,036,945;U.S. Pat. No. 4,331,647; Nisonoff et al., 1960, Arch. Biochem. Biophys.,89:230; Porter, 1959, Biochem. J., 73:119; Edelman et al., 1967, METHODSIN ENZYMOLOGY, page 422 (Academic Press), and Coligan et al. (eds.),1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments or other enzymatic, chemical or genetic techniques also may beused, so long as the fragments bind to the antigen that is recognized bythe intact antibody. For example, Fv fragments comprise an associationof V_(H) and V_(L) chains. This association can be noncovalent, asdescribed in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659.Alternatively, the variable chains may be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. SeeSandhu, 1992, Crit. Rev. Biotech., 12:437.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (sFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains, connected by an oligonucleotidelinker sequence. The structural gene is inserted into an expressionvector that is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingsFvs are well-known in the art. See Whitlow et al., 1991, Methods: ACompanion to Methods in Enzymology 2:97; Bird et al., 1988, Science,242:423; U.S. Pat. No. 4,946,778; Pack et al., 1993, Bio/Technology,11:1271, and Sandhu, 1992, Crit. Rev. Biotech., 12:437.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See Larrick et al., 1991, Methods:A Companion to Methods in Enzymology 2:106; Ritter et al. (eds.), 1995,MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION,pages 166-179 (Cambridge University Press); Birch et al., (eds.), 1995,MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, pages 137-185(Wiley-Liss, Inc.)

Chimeric and Humanized Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of, for example, a human antibody have been replaced by thevariable regions of, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. The affinity of humanized antibodies for a targetmay also be increased by selected modification of the CDR sequences(WO0029584A1). Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993,150:2844.)

Other embodiments may concern non-human primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., WO 91/11465 (1991), and inLosman et al., Int. J. Cancer 46: 310 (1990).

Human Antibodies

In another embodiment, an antibody may be a human monoclonal antibody.Such antibodies may be obtained from transgenic mice that have beenengineered to produce specific human antibodies in response to antigenicchallenge. In this technique, elements of the human heavy and lightchain locus are introduced into strains of mice derived from embryonicstem cell lines that contain targeted disruptions of the endogenousheavy chain and light chain loci. The transgenic mice can synthesizehuman antibodies specific for human antigens, and the mice can be usedto produce human antibody-secreting hybridomas. Methods for obtaininghuman antibodies from transgenic mice are described by Green et al.,Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), andTaylor et al., Int. Immun. 6:579 (1994).

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50; each incorporated herein by reference). Such fully humanantibodies are expected to exhibit even fewer side effects than chimericor humanized antibodies and to function in vivo as essentiallyendogenous human antibodies. In certain embodiments, the claimed methodsand procedures may utilize human antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as HIV infection or AIDS. The advantage toconstructing human antibodies from a diseased individual is that thecirculating antibody repertoire may be biased towards antibodies againstdisease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.) Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.) RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97,incorporated herein by reference). Library construction was performedaccording to Andris-Widhopf et al. (2000, In: Phage Display LaboratoryManual, Barbas et al. (eds), 1^(st) edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporatedherein by reference). The final Fab fragments were digested withrestriction endonucleases and inserted into the bacteriophage genome tomake the phage display library. Such libraries may be screened bystandard phage display methods, such as biopanning. The skilled artisanwill realize that this technique is exemplary only and any known methodfor making and screening human antibodies or antibody fragments by phagedisplay may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. A non-limiting example ofsuch a system is the XENOMOUSE® (e.g., Green et al., 1999, J. Immunol.Methods 231:11-23) from Abgenix (Fremont, Calif.). In the XENOMOUSE® andsimilar animals, the mouse antibody genes have been inactivated andreplaced by functional human antibody genes, while the remainder of themouse immune system remains intact.

The XENOMOUSE® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XENOMOUSE®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XENOMOUSE®are available, each of which is capable of producing a different classof antibody. Such human antibodies may be coupled to other molecules bychemical cross-linking or other known methodologies. Transgenicallyproduced human antibodies have been shown to have therapeutic potential,while retaining the pharmacokinetic properties of normal humanantibodies (Green et al., 1999). The skilled artisan will realize thatthe claimed compositions and methods are not limited to use of theXENOMOUSE® system but may utilize any transgenic animal that has beengenetically engineered to produce human antibodies.

Neutralizing Antibodies

In certain embodiments, neutralizing antibodies or fragments thereofthat are capable of destroying or inhibiting the infectivity and/orvirulence of HIV are preferred. A variety of HIV neutralizing antibodiesare known in the art and any such known antibodies or fragments thereofmay be used, including but not limited to P4/D10, 2G12 (e.g., Joos etal., Antimicrob Agents Chemother 2006, 50:1773-79), 4E10 (Joos et al.,2006), 2F5 (Joos et al., 2006), b12 (e.g., Wu et al., J Virol 2006,80:2585), X5 (Moulard et al., Proc Natl Acad Sci 2002, 99:6913-18) orany combination thereof. Where multispecific antibodies or fragments areused, the skilled artisan will realize that multiple antibodies orfragments that bind to the same or different HIV epitopes may becombined. Although antibodies against the HIV envelope protein (gp120)and/or gp41 are preferred, the skilled artisan will realize that otherHIV target antigens may be utilized to develop antibodies or fragmentsthereof that will target HIV-infected cells. In some cases, antibodiesor fragments that bind to one or more HIV antigens in combination withT-cell antigens (e.g., CD4, CCR5 and/or CXCR4) may be utilized.

Fusion Proteins

Various embodiments may concern fusion proteins. These moleculesgenerally have all or a substantial portion of a peptide, linked at theN- or C-terminus, to all or a portion of a second polypeptide orprotein. For example, fusions may employ leader sequences from otherspecies to permit the recombinant expression of a protein in aheterologous host. Another useful fusion includes the attachment of animmunologically active domain, such as an antibody or fragment, to atherapeutic agent, such as a peptide or protein toxin or enzyme. Yetanother useful form of fusion may include attachment of a moiety of usefor purification, such as the FLAG epitope (Prickett et al., 1989,Biotechniques 7:580-589; Castrucci et al., 1992, J Virol 66:4647-4653).Methods of generating fusion proteins are well known to those of skillin the art. Such proteins may be produced, for example, by chemicalattachment using bifunctional cross-linking reagents, by de novosynthesis of the complete fusion protein, or by attachment of a DNAsequence encoding a first protein or peptide to a DNA sequence encodinga second peptide or protein, followed by expression of the intact fusionprotein.

Bispecific Antibodies

In certain embodiments, bispecific or multispecific antibodies orfragments may be utilized. Such antibodies or fragments will comprise atleast one binding site for an HIV-associated antigen and at least oneother binding site, for example against a carrier molecule conjugated totherapeutic and/or diagnostic agents, a cytokine, a cell surfacereceptor or other antigen.

In general, discrete Y_(R) and V_(L) domains of antibodies produced byrecombinant DNA technology may pair with each other to form a dimer(recombinant Fv fragment) with binding capability (U.S. Pat. No.4,642,334). However, such non-covalently associated molecules are notsufficiently stable under physiological conditions to have any practicaluse. Cognate Y_(R) and V_(L) domains can be joined with a peptide linkerof appropriate composition and length (usually consisting of more than12 amino acid residues) to form a single-chain Fv (scFv) with bindingactivity. Methods of manufacturing scFvs are disclosed in U.S. Pat. No.4,946,778 and U.S. Pat. No. 5,132,405. Reduction of the peptide linkerlength to less than 12 amino acid residues prevents pairing of Y_(H) andY_(L) domains on the same chain and forces pairing of V_(H) and V_(L)domains with complementary domains on other chains, resulting in theformation of functional multimers. Polypeptide chains of V_(H) and V_(L)domains that are joined with linkers between 3 and 12 amino acidresidues form predominantly dimers (termed diabodies). With linkersbetween 0 and 2 amino acid residues, trimers (termed triabodies) andtetramers (termed tetrabodies) are favored, but the exact patterns ofoligomerization appear to depend on the composition as well as theorientation of V-domains (V_(H)-linker-V_(L) or V_(L)-linker-V_(H)), inaddition to the linker length.

Monospecific diabodies, triabodies, and tetrabodies with multiplevalencies have been obtained using peptide linkers consisting of 5 aminoacid residues or less. Bispecific diabodies, which are heterodimers oftwo different scFvs, each scFv consisting of the V_(H) domain from oneantibody connected by a short peptide linker to the V_(L) domain ofanother antibody, have also been made using a dicistronic expressionvector that contains in one cistron a recombinant gene constructcomprising V_(H1)-linker-V_(L2) and in the other cistron a secondrecombinant gene construct comprising V_(H2)-linker-V_(L1) (Holliger, etal. Proc Natl Acad Sci USA. 1993; 90: 6444-6448; Atwell, et al., MolImmunol. 1996; 33:1301-1302; Holliger, et al. Nature Biotechnol. 1997;15: 632-631; Helfrich, et al. Int. J Cancer. 1998; 76: 232-239;Kipriyanov, et al. Int J Cancer. 1998; 77: 763-772; Holliger, et al.Cancer Res. 1999; 59: 2909-2916).

More recently, a tetravalent tandem diabody (termed tandab) with dualspecificity has also been reported (Cochlovius, et al. Cancer Res. 2000;60: 4336-4341). The bispecific tandab is a dimer of two identicalpolypeptides, each containing four variable domains of two differentantibodies (V_(H1), V_(L1), V_(H2), V_(L2)) linked in an orientation tofacilitate the formation of two potential binding sites for each of thetwo different specificities upon self-association.

Methods of manufacturing scFv-based agents of multivalency andmultispecificity by varying the linker length were disclosed in U.S.Pat. No. 5,844,094, U.S. Pat. No. 5,837,242, and WO 98/44001. Methods ofmanufacturing scFv-based agents of multivalency and multispecificity byconstructing two polypeptide chains, one comprising of the V_(H) domainsfrom at least two antibodies and the other the corresponding V_(L)domains were disclosed in U.S. Pat. No. 5,989,830 and U.S. Pat. No.6,239,259. A recombinantly produced bispecific or trispecific antibodyin which the c-termini of CH1 and C_(L) of a Fab are each fused to ascFv derived from the same or different monoclonal antibodies wasdisclosed in U.S. Pat. No. 6,809,185.

Methods for construction and use of bispecific and multispecificantibodies are disclosed, for example, in U.S. Patent ApplicationPublication No. 20050002945, filed Feb. 11, 2004, the entire text ofwhich is incorporated herein by reference. A variety of recombinantmethods can be used to produce bispecific antibodies and antibodyfragments. For example, bispecific antibodies and antibody fragments canbe produced in the milk of transgenic livestock. (See, e.g., Colman, A.,Biochem. Soc. Symp., 63: 141-147, 1998; U.S. Pat. No. 5,827,690, eachincorporated herein by reference.) Two DNA constructs are prepared whichcontain, respectively, DNA segments encoding paired immunoglobulin heavyand light chains. The fragments are cloned into expression vectors whichcontain a promoter sequence that is preferentially expressed in mammaryepithelial cells. Examples include, but are not limited to, promotersfrom rabbit, cow and sheep casein genes, the cow alpha-lactoglobulingene, the sheep beta-lactoglobulin gene and the mouse whey acid proteingene. Preferably, the inserted fragment is flanked on its 3′ side bycognate genomic sequences from a mammary-specific gene. This provides apolyadenylation site and transcript-stabilizing sequences. Theexpression cassettes are coinjected into the pronuclei of fertilized,mammalian eggs, which are then implanted into the uterus of a recipientfemale and allowed to gestate. After birth, the progeny are screened forthe presence of both transgenes by Southern analysis. In order for theantibody to be present, both heavy and light chain genes must beexpressed concurrently in the same cell. Milk from transgenic females isanalyzed for the presence and functionality of the antibody or antibodyfragment using standard immunological methods known in the art. Theantibody can be purified from the milk using standard methods known inthe art.

Pre-Targeting

One strategy for use of bispecific antibodies includes pretargetingmethodologies, in which an effector molecule is administered to asubject after a bispecific antibody has been administered. Thebispecific antibody, which would include a binding site for an HIVantigen and one for a carrier conjugated to one or more effectormolecules, localizes to the diseased tissue and increases thespecificity of localization of the effector to the diseased tissue (U.S.Patent Application No. 20050002945). Because the effector molecule maybe cleared from circulation much more rapidly than the bispecificantibody, normal tissues may have a decreased exposure to the effectormolecule when a pretargeting strategy is used than when the effectormolecule is directly linked to the disease targeting antibody.

Pretargeting methods have been developed to increase thetarget:background ratios of detection or therapeutic agents. Examples ofpre-targeting and biotin/avidin approaches are described, for example,in Goodwin et al., U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl.Med. 29:226, 1988; Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehret al., J. Nucl. Med. 29:728, 1988; Klibanov et al., J. Nucl. Med.29:1951, 1988; Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos etal., J. Nucl. Med. 31:1791, 1990; Schechter et al., Int. J. Cancer48:167, 1991; Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli etal., Nucl. Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickneyet al., Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119,1991; U.S. Pat. No. 6,077,499; U.S. Ser. No. 09/597,580; U.S. Ser. No.10/361,026; U.S. Ser. No. 09/337,756; U.S. Ser. No. 09/823,746; U.S.Ser. No. 10/116,116; U.S. Ser. No. 09/382,186; U.S. Ser. No. 10/150,654;U.S. Pat. No. 6,090,381; U.S. Pat. No. 6,472,511; U.S. Ser. No.10/114,315; U.S. Provisional Application No. 60/386,411; U.S.Provisional Application No. 60/345,641; U.S. Provisional Application No.60/3328,835; U.S. Provisional Application No. 60/426,379; U.S. Ser. No.09/823,746; U.S. Ser. No. 09/337,756; and U.S. Provisional ApplicationNo. 60/342,103, all of which are incorporated herein by reference.

In certain embodiments, bispecific antibodies and targetable constructsmay be of use in treating and/or imaging diseased tissues, for exampleusing the methods described in U.S. Pat. Nos. 6,126,916; 6,077,499;6,010,680; 5,776,095; 5,776,094; 5,776,093; 5,772,981; 5,753,206;5,746,996; 5,697,902; 5,328,679; 5,128,119; 5,101,827; and 4,735,210,each incorporated herein by reference. Additional methods are describedin U.S. application Ser. No. 09/337,756 filed Jun. 22, 1999 and in U.S.application Ser. No. 09/823,746, filed Apr. 3, 2001.

Dock and Lock (DNL)

In certain embodiments, bispecific antibodies or fragments, orconjugates of antibodies or fragments, may be assembled using atechnology known as dock and lock (DNL). Further details of DNLtechnology may be found in U.S. patent application Ser. No. 11/389,358,filed Mar. 24, 2005; Ser. No. 11/391,584, filed Mar. 28, 2006; Ser. No.11/478,021, filed Jun. 29, 2006; Ser. No. 11/633,729, filed Jun. 21,2007; and U.S. Provisional Patent Application Ser. No. 60/864,530, filedNov. 6, 2006, the text of each of which is incorporated herein byreference in its entirety.

To summarize, DNL technology involves targeting binding between two ormore complementary sequences, such as a dimerization and docking domain(DDD) of, for example, the regulatory subunits of c-AMP-dependentprotein kinas A, and the anchoring domain found in various A-kinaseanchoring proteins (AKAPs) that mediates association with the R subunitsof PKA. However, the skilled artisan will realize that otherdimerization and docking domains and anchoring domains are known and anysuch known domains may be used within the scope of the claimed subjectmatter. Other exemplary 4-helix bundle type DDD domains may be obtainedfrom p53, DCoH (pterin 4 alpha carbinolamine dehydratase/dimerizationcofactor of hepatocyte nuclear factor 1 alpha (TCF1)) and HNF-1(hepatocyte nuclear factor 1). Other AD sequences of potential use maybe found in Patent Application Serial No. US20003/0232420A1, the entiretext of which is incorporated herein by reference.

These complementary binding pairs may be covalently attached to variousfunctional units, such as antibodies or antibody fragments, ortherapeutic or diagnostic agents, forming therapeutic or diagnosticcomplexes of determined specificity and activity. The skilled artisanwill realize that there are a multiplicity of ways in which suchcomplexes could be formed, for example by incorporating AD or DDDsequences into fusion proteins comprising a Fab, Fab′, IgG, scFc orother antibody or fragment with known binding specificity. For example,a bispecific complex may be formed by incorporating AD and DDD sequencesinto antibodies or fragments specific for two different target antigens.Alternatively, an antibody or fragment attached to a DDD or AD moietymay be combined with a therapeutic protein or peptide, such as ahormone, enzyme, ribonuclease, onconase or other agent that is attachedto a complementary AD or DDD moiety. A number of such potentialcomplexes are described in the patent applications listed above and anyof such known complexes may be utilized.

Conjugation of Therapeutic or Diagnostic Agents to anti-HIV Antibodies

In various embodiments, therapeutic agents may be conjugated to anti-HIVantibodies, fragments or other targeting molecules for delivery to HIVinfected cells. Therapeutic agents of use may comprise one or more ofaplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin,irinotecan (CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide,cytarabine, dacarbazine, dactinomycin, daunomycin glucuronide,daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicinglucuronide, estramustine, etoposide, etoposide glucuronide, etoposidephosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO),fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine,hydroxyurea, idarubicin, ifosfamide, L-asparaginase, leucovorin,lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, phenylbutyrate, procarbazine, pentostatin, PSI-341, semustine, streptozocin,taxanes, thioguanine, thiotepa, teniposide, topotecan, uracil mustard,velcade, vinblastine, vinorelbine, vincristine, ricin, abrin,ribonuclease, onconase, rapLR1, DNase I, Staphylococcal enterotoxin-A,pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonasexotoxin, Pseudomonas endotoxin, an antisense oligonucleotide, aninterference RNA, or a combination thereof.

Additional moieties can be conjugated to the HIV targeting moleculesdescribed herein. For example, drugs, toxins, radioactive compounds,enzymes, hormones, cytotoxic proteins, chelates, cytokines, and otherfunctional agents may be conjugated to the HIV targeting molecules.Conjugation can be via, for example, covalent attachments to amino acidresidues containing amine, carboxyl, thiol or hydroxyl groups in theirside-chains. Various conventional linkers may be used for this purpose,for example, diisocyanates, diisothiocyanates, bis(hydroxysuccinimide)esters, carbodiimides, maleimide-hydroxysuccinimide esters,glutaraldehyde and the like. Conjugation of agents to the HIV targetingmolecules preferably does not significantly affect the binding activityor specificity compared to the unmodified structures. In addition,cytotoxic and/or virostatic agents may be first coupled to a polymericcarrier, which is then conjugated to a HIV targeting molecule. For thismethod, see Ryser et al., Proc. Natl. Acad. Sci. USA, 75:3867-3870,1978, U.S. Pat. No. 4,699,784, and U.S. Pat. No. 4,046,722, which areincorporated herein by reference.

The conjugates described herein can be prepared by methods known forlinking antibodies with lipids, carbohydrates, proteins, radionuclides,or other atoms and molecules. For example, the HIV targeting moleculesdescribed herein can be linked to one or more of the carriers describedherein (e.g., lipids, polymers, liposomes, micelles, or nanoparticles)to form a conjugate, which can then incorporate a therapeutic ordiagnostic agent either covalently, non-covalently, or otherwise.Alternatively, any of the HIV targeting molecules described herein canbe conjugated directly with one or more therapeutic or diagnostic agentsdescribed herein.

For example, a HIV targeting molecule can be radiolabeled with ¹³¹I andconjugated to a lipid, such that the resulting conjugate can form aliposome. The liposome may incorporate one or more therapeutic (e.g., adrug such as FUdR-dO) or diagnostic agents. The formation of liposomesand micelles is known in the art. See, e.g., Wrobel and Collins,Biochimica et Biophysica Acta (1995), 1235: 296-304; Lundberg et al., J.Pharm. Pharmacol. (1999), 51:1099-1105; Lundberg et al., Int. J. Pharm.(2000), 205:101-108; Lundberg, J. Pharm. Sci. (1994), 83:72-75; Xu etal., Molec. Cancer Ther. (2002), 1:337-346; Torchilin et al., Proc.Nat'l. Acad. Sci., U.S.A. (2003), 100:6039-6044; U.S. Pat. No.5,565,215; U.S. Pat. No. 6,379,698; and U.S. 2003/0082154.

Nanoparticles or nanocapsules formed from polymers, silica, or metals,which are useful for drug delivery or imaging, have been described aswell. See, e.g., West et al., Applications of Nanotechnology toBiotechnology (2000), 11:215-217; U.S. Pat. No. 5,620,708; U.S. Pat. No.5,702,727; and U.S. Pat. No. 6,530,944. The conjugation of antibodies orbinding molecules to liposomes to form a targeted carrier fortherapeutic or diagnostic agents has been described. See, e.g., Bendas,Biodrugs (2001), 15:215-224; Xu et al., Mol. Cancer Ther (2002),1:337-346; Torchilin et al., Proc. Nat'l. Acad. Sci. U.S.A (2003),100:6039-6044; Bally, et al., J. Liposome Res. (1998), 8:299-335;Lundberg, Int. J. Pharm. (1994), 109:73-81; Lundberg, J. Pharm.Pharmacol. (1997), 49:16-21; Lundberg, Anti-cancer Drug Design (1998),13: 453-461. See also U.S. Pat. No. 6,306,393; U.S. Ser. No. 10/350,096;U.S. Ser. No. 09/590,284, and U.S. Ser. No. 60/138,284, filed Jun. 9,1999. All these references are incorporated herein by reference.

A wide variety of diagnostic and therapeutic agents can beadvantageously used to form the conjugates of the HIV targetingmolecules, or may be linked to haptens that bind to a recognition siteon the HIV targeting molecules. Diagnostic agents may includeradioisotopes, enhancing agents for use in MRI or contrast agents forultrasound imaging, and fluorescent compounds. Many appropriate imagingagents are known in the art, as are methods for their attachment toproteins or peptides (see, e.g., U.S. Pat. Nos. 5,021,236 and 4,472,509,both incorporated herein by reference). Certain attachment methodsinvolve the use of a metal chelate complex employing, for example, anorganic chelating agent such a DTPA attached to the protein or peptide(U.S. Pat. No. 4,472,509).

In order to load a HIV targeting molecule with radioactive metals orparamagnetic ions, it may be necessary to first react it with a carrierto which multiple copies of a chelating group for binding theradioactive metals or paramagnetic ions have been attached. Such acarrier can be a polylysine, polysaccharide, or a derivatized orderivatizable polymeric substance having pendant groups to which can bebound chelating groups such as, e.g., ethylenediaminetetraacetic acid(EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins,polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and thelike known to be useful for this purpose. Carriers containing chelatesare coupled to the HIV targeting molecule using standard chemistries ina way to minimize aggregation and loss of immunoreactivity.

Other, more unusual, methods and reagents that may be applied forpreparing such conjugates are disclosed in U.S. Pat. No. 4,824,659,which is incorporated herein in its entirety by reference. Particularlyuseful metal-chelate combinations include 2-benzyl-DTPA and itsmonomethyl and cyclohexyl analogs, used with diagnostic isotopes in thegeneral energy range of 60 to 4,000 keV. Some useful diagnostic nuclidesmay include ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁴Tc,^(94m)Tc, ^(99m)Tc, or ¹¹¹In. The same chelates complexed withnon-radioactive metals, such as manganese, iron and gadolinium, areuseful for MRI, when used along with the HIV targeting molecules andcarriers described herein. Macrocyclic chelates such as NOTA, DOTA, andTETA are of use with a variety of metals and radiometals, mostparticularly with radionuclides of gallium, yttrium and copper,respectively. Such metal-chelate complexes can be made very stable bytailoring the ring size to the metal of interest. Other ring-typechelates, such as macrocyclic polyethers for complexing ²²³Ra, may beused.

Therapeutic agents include, for example, chemotherapeutic drugs such asvinca alkaloids, anthracyclines, epidophyllotoxins, taxanes,antimetabolites, alkylating agents, antibiotics, Cox-2 inhibitors,antimitotics, antiangiogenic and proapoptotic agents, particularlydoxorubicin, methotrexate, taxol, CPT-11, camptothecans, and others fromthese and other classes of cytotoxic agents. Other cytotoxic agentsinclude nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes,folic acid analogs, pyrimidine analogs, purine analogs, platinumcoordination complexes, and the like. Suitable cytotoxic agents aredescribed in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (MackPublishing Co. 1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICALBASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as wellas revised editions of these publications. Other suitable cytotoxicagents, such as experimental drugs, are known to those of skill in theart, and may be conjugated to the HIV targeting molecules describedherein using methods that are known in the art.

Another class of therapeutic agents consists of radionuclides that emitα-particles (such as ²¹²Pb, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²³Ra, ²²⁵Ac,β-particles (such as ³²P, ³³P, ⁴⁷Sc, ⁶⁷Cu, ⁶⁷Ga, ⁸⁹Sr, ⁹⁰Y, ¹¹¹Ag, ¹²⁵I,¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁶Dy, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re), orAuger electrons (such as ¹¹¹In, ¹²⁵I, ⁶⁷Ga, ¹⁹¹Os, ^(193m)Pt, ^(195m)Pt,^(195m)Hg). The HIV targeting molecules may be labeled with one or moreof the above radionuclides using methods as described for the diagnosticagents.

In certain embodiments, the therapeutic agents of use may comprise oneor more aggresome inhibitors. Aggresomes are large intracellularcomplexes that were thought to form in response to misfolded protein(see, e.g., Heath et al., J. Cell Biol. 153:449-55, 2001; Johnstone etal., J. Cell Biol. 143:1883-98, 1998; Wileman, Science 312:875-78,2006). More recently, it has been suggested that aggresomes may functionin the assembly of viral particles (Heath et al., 2001; Wileman, 2006).Aggresome inhibitors may therefore function to block or inhibit theformation of new infectious viral particles from cells infected with HIVor other viruses. A variety of aggresome inhibitors are known, such asALLN, nocodazole, colchicine and vinblastine (Johnston et al., 1998),other microtubule inhibitors (Gerdes and Katsanis, Hum. Molec. Genet.14:R291-300, 2005); bortezomib (Velcade) (Catley et al., Blood108:3441-49, 2006), tubacin, histone deacetylase inhibitors (Corcoran etal., Curr. Biol. 14:488-92, 2004), and any such known aggresomeinhibitor may be used.

In various embodiments, one or more immunomodulators may be conjugatedto an anti-HIV antibody or fragment for administration to a patient, oralternatively may be co-administered to the patient. As used herein, theterm “immunomodulator” includes cytokines, stem cell growth factors,lymphotoxins and hematopoietic factors, such as interleukins, colonystimulating factors, interferons (e.g., interferons-α, -β and -γ) andthe stem cell growth factor designated “S1 factor.” Examples of suitableimmunomodulator moieties include IL-2, IL-6, IL-10, IL-12, IL-18, IL-21,interferon-gamma, TNF-alpha, and the like.

The term “cytokine” is a generic term for proteins or peptides releasedby one cell population which act on another cell as intercellularmediators. As used broadly herein, examples of cytokines includelymphokines, monokines, growth factors and traditional polypeptidehormones. Included among the cytokines are growth hormones such as humangrowth hormone, N-methionyl human growth hormone, and bovine growthhormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);hepatic growth factor; prostaglandin, fibroblast growth factor;prolactin; placental lactogen, OB protein; tumor necrosis factor-α and-β; mullerian-inhibiting substance; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-21, G-CSF, GM-CSF, M-CSF, EPO, kit-ligand or FLT-3,angiostatin, thrombospondin, endostatin, tumor necrosis factor and LT.As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

Chemokines generally act as chemoattractants to recruit immune effectorcells to the site of chemokine expression. It may be advantageous toexpress a particular chemokine gene in combination with, for example, acytokine gene, to enhance the recruitment of other immune systemcomponents to a site of treatment. Chemokines include, but are notlimited to, RANTES, MCAF, MIP1-alpha, MIP1-Beta, and IP-10. The skilledartisan will recognize that certain cytokines are also known to havechemoattractant effects and could also be classified under the termchemokines. Similarly, the terms immunomodulator and cytokine overlap intheir respective members.

A suitable peptide containing a detectable label (e.g., a fluorescentmolecule), or a virostatic and/or cytotoxic agent, (e.g., aradioiodine), can be covalently, non-covalently, or otherwise associatedwith the HIV targeting molecules. For example, a therapeutically usefulconjugate can be obtained by incorporating a photoactive agent or dyeonto the HIV targeting molecules. Fluorescent compositions, such asfluorochrome, and other chromogens, or dyes, such as porphyrinssensitive to visible light, have been used to detect and to treatlesions by directing the suitable light to the lesion. In therapy, thishas been termed photoradiation, phototherapy, or photodynamic therapy.See Joni et al. (eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHERDISEASES (Libreria Progetto 1985); van den Bergh, Chem. Britain (1986),22:430. Moreover, monoclonal antibodies have been coupled withphotoactivated dyes for achieving phototherapy. See Mew et al., J.Immunol. (1983), 130:1473; idem., Cancer Res. (1985), 45:4380; Oseroffet al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem., Photochem.Photobiol. (1987), 46:83; Hasan et al., Prog. Clin. Biol. Res. (1989),288:471; Tatsuta et al., Lasers Surg. Med. (1989), 9:422; Pelegrin etal., Cancer (1991), 67:2529.

Aptamers

In alternative embodiments, the disclosed compositions and methods mayutilize alternative forms of binding molecules to antibodies or antibodyfragments, such as aptamers. Aptamers are typically syntheticoligonucleotides that can adopt three-dimensional conformations thatprovide antibody-like binding affinities and specificities for selectedtarget molecules. Methods of constructing and determining the bindingcharacteristics of aptamers are well known in the art. For example, suchtechniques are described in U.S. Pat. Nos. 5,582,981, 5,595,877 and5,637,459, each incorporated herein by reference.

Aptamers may be prepared by any known method, including synthetic,recombinant, and purification methods, and may be used alone or incombination with other ligands specific for the same target. In general,a minimum of approximately 3 nucleotides, preferably at least 5nucleotides, are necessary to effect specific binding. Aptamers ofsequences shorter than 10 bases may be feasible, although aptamers of10, 20, 30 or 40 nucleotides may be preferred.

Aptamers need to contain the sequence that confers binding specificity,but may be extended with flanking regions and otherwise derivatized. Inpreferred embodiments, the binding sequences of aptamers may be flankedby primer-binding sequences, facilitating the amplification of theaptamers by PCR or other amplification techniques. In a furtherembodiment, the flanking sequence may comprise a specific sequence thatpreferentially recognizes or binds a moiety to enhance theimmobilization of the aptamer to a substrate.

Aptamers may be isolated, sequenced, and/or amplified or synthesized asconventional DNA or RNA molecules. Alternatively, aptamers of interestmay comprise modified oligomers. Any of the hydroxyl groups ordinarilypresent in aptamers may be replaced by phosphonate groups, phosphategroups, protected by a standard protecting group, or activated toprepare additional linkages to other nucleotides, or may be conjugatedto solid supports. One or more phosphodiester linkages may be replacedby alternative linking groups, such as P(O)O replaced by P(O)S, P(O)NR₂,P(O)R, P(O)OR′, CO, or CNR₂, wherein R is H or alkyl (1-20C) and R′ isalkyl (1-20C); in addition, this group may be attached to adjacentnucleotides through O or S. Not all linkages in an oligomer need to beidentical.

Methods for preparation and screening of aptamers that bind toparticular targets of interest are well known, for example U.S. Pat. No.5,475,096 and U.S. Pat. No. 5,270,163, each incorporated by reference.The technique generally involves selection from a mixture of candidateaptamers and step-wise iterations of binding, separation of bound fromunbound aptamers and amplification. Because only a small number ofsequences (possibly only one molecule of aptamer) corresponding to thehighest affinity aptamers exist in the mixture, it is generallydesirable to set the partitioning criteria so that a significant amountof aptamers in the mixture (approximately 5-50%) is retained duringseparation. Each cycle results in an enrichment of aptamers with highaffinity for the target. Repetition for between three to six selectionand amplification cycles may be used to generate aptamers that bind withhigh affinity and specificity to the target. Aptamers may be conjugatedto therapeutic agents by standard nucleic acid labeling techniques wellknown in the art.

Avimers

In certain embodiments, the disclosed compositions or methods mayutilize one or more avimer sequences. Avimers are a class of bindingproteins somewhat similar to antibodies in their affinities andspecificities for various target molecules. They were developed fromhuman extracellular receptor domains by in vitro exon shuffling andphage display. (Silverman et al., 2005, Nat. Biotechnol. 23:1493-94;Silverman et al., 2006, Nat. Biotechnol. 24:220.) The resultingmultidomain proteins may comprise multiple independent binding domainsthat may exhibit improved affinity (in some cases sub-nanomolar) andspecificity compared with single-epitope binding proteins. (Id.) Invarious embodiments, avimers may be attached to therapeutic agents foruse in the claimed methods and compositions using standard proteincross-linking or labeling techniques discussed herein. Additionaldetails concerning methods of construction and use of avimers aredisclosed, for example, in U.S. Patent Application Publication Nos.20040175756, 20050048512, 20050053973, 20050089932 and 20050221384, theExamples section of each of which is incorporated herein by reference.

Formulation and Administration

The HIV targeting molecules, including their conjugates, may be furtherformulated to obtain compositions that include one or morepharmaceutically suitable excipients, one or more additionalingredients, or some combination of these. These can be accomplished byknown methods to prepare pharmaceutically useful dosages, whereby theactive ingredients (i.e., the HIV targeting molecules or conjugates),are combined in a mixture with one or more pharmaceutically suitableexcipients. Sterile phosphate-buffered saline is one example of apharmaceutically suitable excipient. Other suitable excipients are wellknown to those in the art. See, e.g., Ansel et al., PHARMACEUTICALDOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18thEdition (Mack Publishing Company 1990), and revised editions thereof.

One route for administration of the compositions described herein isparenteral injection. In parenteral administration, the compositionswill be formulated in a unit dosage injectable form such as a solution,suspension or emulsion, in association with a pharmaceuticallyacceptable excipient. Such excipients are inherently nontoxic andnontherapeutic. Examples of such excipients are saline, Ringer'ssolution, dextrose solution and Hank's solution. Nonaqueous excipientssuch as fixed oils and ethyl oleate may also be used. A preferredexcipient is 5% dextrose in saline. The excipient may contain minoramounts of additives such as substances that enhance isotonicity andchemical stability, including buffers and preservatives. Other methodsof administration, including oral administration, are also contemplated.

Formulated compositions comprising HIV targeting molecules can be usedfor intravenous administration via, for example, bolus injection orcontinuous infusion. Compositions for injection can be presented in unitdosage form, e.g., in ampules or in multi-dose containers, with an addedpreservative. Compositions can also take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the compositions can be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compositions may be administered in solution. The pH of the solutionshould be in the range of pH 5 to 9.5, preferably pH 6.5 to 7.5. Theformulation thereof should be in a solution having a suitablepharmaceutically acceptable buffer such as phosphate,tris(hydroxymethyl)aminomethane-HCl or citrate and the like. Bufferconcentrations should be in the range of 1 to 100 mM. The formulatedsolution may also contain a salt, such as sodium chloride or potassiumchloride in a concentration of 50 to 150 mM. An effective amount of astabilizing agent such as glycerol, albumin, a globulin, a detergent, agelatin, a protamine or a salt of protamine may also be included.Systemic administration of the formulated composition is typically madeevery two to three days or once a week if a humanized form of theantibody is used as a template for the HIV targeting molecules.Alternatively, daily administration is useful. Usually administration isby either intramuscular injection or intravascular infusion.

The compositions may be administered to a mammal subcutaneously or byother parenteral routes. Moreover, the administration may be bycontinuous infusion or by single or multiple boluses. Methods useful forthe antibodies or immunoconjugates can be applied to the compositionsdescribed herein. In general, the dosage of an administeredimmunoconjugate, fusion protein or naked antibody for humans will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of the activeingredient that is in the range of from about 1 mg/kg to 20 mg/kg as asingle intravenous infusion, although a lower or higher dosage also maybe administered as circumstances dictate. This dosage may be repeated asneeded, for example, once per week for 4-10 weeks, preferably once perweek for 8 weeks, and more preferably, once per week for 4 weeks. It mayalso be given less frequently, such as every other week for severalmonths. The dosage may be given through various parenteral routes, withappropriate adjustment of the dose and schedule.

Pharmaceutical methods employed to control the duration of action ofimmunoconjugates or antibodies may be applied to the formulatedcompositions described herein. Control release preparations can beachieved through the use of biocompatible polymers to complex or adsorbthe immunoconjugate or naked antibody, for example, matrices ofpoly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. See Sherwood et al.,Bio/Technology (1992), 10: 1446. The rate of release of animmunoconjugate or antibody from such a matrix depends upon themolecular weight of the immunoconjugate or antibody, the amount ofimmunoconjugate, antibody within the matrix, and the size of dispersedparticles. See Saltzman et al., Biophys. J (1989), 55: 163; Sherwood etal., supra. Other solid dosage forms are described in Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

For purposes of therapy, the composition is administered to a mammal ina therapeutically effective amount. A suitable subject for thetherapeutic and diagnostic methods disclosed herein is usually a human,although a non-human animal subject is also contemplated.

The compositions may be administered by aerosol to achieve localizeddelivery to the lungs. Either an aqueous aerosol or a nonaqueous (e.g.,fluorocarbon propellent) suspension could be used. Sonic nebulizerspreferably are used in preparing aerosols to minimize exposing the HIVtargeting molecule in the compositions to shear, which can result in itsdegradation and loss of activity.

A HIV targeting molecule linked to a radionuclide may be effective fortherapy. After it has been determined that the HIV targeting molecule islocalized at one or more infectious sites in a subject, higher doses ofthe labeled composition, generally from 20 mCi to 150 mCi per dose for¹³¹I, 5 mCi to 30 mCi per dose for ⁹⁰Y, or 5 mCi to 20 mCi per dose of¹⁸⁶Re, each based on a 70 kg patient weight, are injected. Injection maybe intravenous, intraarterial, intralymphatic, intrathecal, orintracavitary (i.e., parenterally), and may be repeated. It may beadvantageous for some therapies to administer multiple, divided doses,thus providing higher toxic doses without usually effecting aproportional increase in radiation of normal tissues.

In still other embodiments, anti-HIV antibodies or fragments may bemodified for oral or inhalational administration by conjugation tocertain proteins, such as the Fc region of IgG1 (see Examples 3-7).Methods for preparation and use of peptide-Fc conjugates are disclosed,for example, in Low et al. (2005, Hum. Reprod. 20:1805-13) and Dumont etal. (2005, J. Aerosol. Med. 18:294-303), each incorporated herein byreference. Low et al. (2005) disclose the conjugation of the alpha andbeta subunits of FSH to the Fc region of IgG1 in single chain orheterodimer form, using recombinant expression in CHO cells. The Fcconjugated peptides were absorbed through epithelial cells in the lungor intestine by the neonatal Fc receptor mediated transport system. TheFc conjugated peptides exhibited improved stability and absorption invivo compared to the native peptides. It was also observed that theheterodimer conjugate was more active than the single chain form. Largerproteins, such as erythropoietin, may also be effectively delivered byinhalation using Fc conjugation (Dumont et al., 2005).

Uses for Treatment and Diagnosis: Applications Not InvolvingPretargeting

The use of radioactive and non-radioactive diagnostic agents, which arelinked to the HIV targeting molecules, is contemplated. Suitablenon-radioactive diagnostic agents are those used for magnetic resonanceimaging (MRI), computed tomography (CT) or ultrasound. MRI agentsinclude, for example, non-radioactive metals, such as manganese, ironand gadolinium, which are complexed with suitable chelates such as2-benzyl-DTPA and its monomethyl and cyclohexyl analogs. See U.S. Ser.No. 09/921,290 filed on Oct. 10, 2001, which is incorporated in itsentirety by reference.

The HIV targeting molecules may be labeled with a radioisotope usefulfor diagnostic imaging. Suitable radioisotopes may include those in theenergy range of 60 to 4,000 KeV, or more specifically, ¹⁸F, ⁵²Fe, ⁶²Cu,⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr ^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ⁴⁵Ti, ¹¹¹In,¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ¹⁷⁷Lu, ³²P, ¹⁸⁸Re, and the like, or acombination thereof. See, e.g., U.S. Patent Application entitled“Labeling Targeting Agents with Gallium-68”—Inventors G. L. Griffithsand W. J. McBride, and U.S. Provisional Application No. 60/342,104,which discloses positron emitters, such as ¹⁸F, ⁶⁸Ga, ^(94m)Tc, and thelike, for imaging purposes; (incorporated herein by reference).Detection can be achieved, for example, by single photon emissioncomputed tomography (SPECT), or positron emission tomography (PET).

In another embodiment the HIV targeting molecules may be labeled withone or more radioactive isotopes useful for killing HIV-infected cells,which include β-emitters (such as ³²P, ³³P, ⁴⁷S, ⁶⁷Cu, ⁶⁷Ga, ⁸⁹Sr, ⁹⁰Y,¹¹¹Ag, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁶Dy, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ¹⁸⁹Re), Auger electron emitters (such as ¹¹¹In, ¹²⁵I, ⁶⁷Ga,¹⁹¹Os, ^(193m)Pt, ¹⁹⁵Pt, ^(195m)Hg, α-emitters (such as ²¹²Pb, ²¹²Bi,²¹³Bi, ²¹¹At, ²²³Ra, ²²⁵Ac), or a combination thereof.

The HIV targeting molecules may be used for MRI by linking to one ormore image enhancing agents, which may include complexes of metalsselected from the group consisting of chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) and erbium (III).Similarly, the HIV targeting molecules may be used for ultrasoundimaging by linking to one or more image enhancing agents currently onthe market. U.S. Pat. No. 6,331,175 describes MRI technique and thepreparation of antibodies conjugated to an MRI enhancing agent and isincorporated in its entirety by reference.

A functional protein, such as a toxin, may be present in the HIVtargeting molecules in several ways. For example, a functional proteinmay be directly attached to an anti-HIV antibody or fragment as a fusionprotein, using standard molecular biology techniques. Alternativelyfunctional proteins may be covalently conjugated to anti-HIV antibodiesor fragments by known chemical cross-linking methods. Toxins that may beused in this regard include ricin, abrin, ribonuclease (RNase), DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. (See,e.g., Pastan et al., Cell (1986), 47:641; Goldenberg, C A—A CancerJournal for Clinicians (1994), 44:43; Sharkey and Goldenberg, C A CancerJ. Clin. 56:226 (2006), each incorporated herein by reference.)Additional toxins suitable for use herein are known to those of skill inthe art and are disclosed in U.S. Pat. No. 6,077,499, which isincorporated in its entirety by reference. Other functional proteins ofinterest include various cytokines, enzymes, and fluorescent proteins.

Uses for Treatment and Diagnosis: Applications Involving Pretargeting

Pretargeting is a multistep process originally developed to resolve theslow blood clearance of directly targeting antibodies, which contributesto undesirable toxicity to normal tissues, in particular, bone marrow.With pretargeting, a radionuclide or other therapeutic agent is attachedto a small compound that is cleared within minutes from the blood. Thepretargeting agent, which is capable of recognizing the smallradiolabeled compound in addition to the target antigen, is administeredfirst, and the radiolabeled compound is administered at a later timewhen the pretargeting agent is sufficiently cleared from the blood.

A pretargeting method of treating or diagnosing a disease or disorder ina subject is provided by: (1) administering to the subject a bispecificHIV-targeting molecule as described above, where at least one antigenbinding site is directed against an HIV marker, and the other antigenbinding site is directed to a targetable construct containing a bivalenthapten; (2) optionally administering to the subject a clearingcomposition, and allowing the composition to clear the binding structurefrom circulation; and (3) administering to the subject the targetableconstruct containing a bivalent hapten, where the targetable constructfurther contains one or more chelated or chemically bound therapeutic ordiagnostic agents. A variety of clearing agents and methods are known inthe art and may be used for pretargeting, such as anti-idiotypicantibodies that complex with an HIV-targeting antibody or attachment oftags or labels (e.g., monosaccharides) to an HIV-targeting antibody thatmay be complexed with other agents (such as a monosaccharide-bindingantibody). Use of avidin-biotin binding interactions for clearingmethods are also known in the art.

Also provided is a method of antibody dependent enzyme prodrug therapy(ADEPT) by (1) administering to a patient an HIV targeting molecule asabove, where the structure contains a covalently attached enzyme capableof activating a prodrug, (2) optionally administering to the subject aclearing composition, and allowing the composition to clear the bindingstructure from circulation, and (3) administering the prodrug to thepatient.

IMP 411 Targetable Construct

In preferred embodiments, a targetable construct may comprise one ormore histamine-succinyl-glycine (HSG) moieties and an antigen-bindingsite may be associated with an HSG-binding antibody or fragment, such asthe 679 antibody or fragment. (see, e.g., U.S. Pat. No. 6,962,702,incorporated herein by reference). The targetable construct may beconjugated to any known diagnostic and/or therapeutic agent, using knownpretargeting techniques (e.g., U.S. Pat. No. 6,962,702). An exemplarytargetable construct, known as IMP 411, that comprises two HSG moietieshas the formulaDOTA-D-Cys(3-SP-Gly-20-O-SN38)-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂.IMP 411 may be linked, for example, to SN38 chemotherapeutic agents, asindicated in the formula above. Alternatively, other therapeutic ordiagnostic agents may be incorporated into or conjugated to the samepeptide backbone.

Additional Uses

In certain embodiments, HIV targeting molecules may be of use intreating and/or imaging HIV-infected tissues, for example using themethods described in U.S. Pat. Nos. 6,126,916; 6,077,499; 6,010,680;5,776,095; 5,776,094; 5,776,093; 5,772,981; 5,753,206; 5,746,996;5,697,902; 5,328,679; 5,128,119; 5,101,827; and 4,735,210, eachincorporated herein by reference. Additional methods are described inU.S. application Ser. No. 09/337,756 filed Jun. 22, 1999 and in U.S.application Ser. No. 09/823,746, filed Apr. 3, 2001. Such imaging can beconducted by direct labeling of the HIV targeting molecule, or by apretargeted imaging method, as described in Goldenberg et al, “AntibodyPretargeting Advances Cancer Radioimmunodetection and Radiotherapy,” (J.Clin. Oncol., 2006 24:823-34), see also U.S. Patent Publication Nos.20050002945, 20040018557, 20030148409 and 20050014207, each incorporatedherein by reference.

In some embodiments, the HIV targeting molecules disclosed and claimedherein may be of use in radionuclide therapy or radioimmunotherapymethods (see, e.g., Govindan et al., 2005, Technology in Cancer Research& Treatment, 4:375-91; Sharkey and Goldenberg, 2005, J. Nucl. Med.46:115S-127S; Goldenberg et al., 2006, J. Clin. Oncol. 24:823-34, eachincorporated herein by reference.)

In another embodiment, a radiosensitizer can be used in combination witha naked or conjugated HIV targeting molecule, antibody or antibodyfragment. For example, the radiosensitizer can be used in combinationwith a radiolabeled HIV targeting molecule. The addition of theradiosensitizer can result in enhanced efficacy when compared totreatment with the radiolabeled HIV targeting molecule alone.Radiosensitizers are described in D. M. Goldenberg (ed.), CANCER THERAPYWITH RADIOLABELED ANTIBODIES, CRC Press (1995), which is incorporatedherein by reference.

The HIV targeting molecule, for use in any of the claimed methods, maybe associated or administered with cytokines and immune modulators.These cytokines and immune modulators, include, at least, interferons ofalpha, beta and gamma, and colony stimulating factors. However, otherimmune modulators are known and may be used, such as IL-1, IL-2, IL-3,IL-6, IL-10, IL-12, IL-18, TNF-α, stem cell growth factors,lymphotoxins, erythropoietin, thrombopoietin, G-CSF, GM-CSF, S1 factoror a combination thereof.

Alternative embodiments concern methods for the intravascularidentification of HIV-infected tissues, in a subject by administering aneffective amount of a HIV targeting molecule and a targetable construct.The HIV targeting molecule comprises at least one ABS that specificallybinds to an HIV antigen, and at least one ABS that specifically binds atargetable construct.

Kits

Some embodiments concern kits for practicing the claimed methods. Thekit may include an HIV targeting molecule. The targeting molecule may belabeled by any of the agents described above. Further, the targetingmolecule may be unlabeled but the kit may comprise labeling reagents tolabel the targeting molecule. The labeling reagents, if included, maycontain the label and a crosslinker. The kit may also contain a HIVtargeting molecule comprising at least one antibody specific for an HIVantigen and at least one antibody specific for a carrier. The kit mayoptionally contain a clearing composition to remove HIV targetingmolecule from circulation.

The kit components may be packaged together or separated into two ormore separate containers. In some embodiments, the containers may bevials that contain sterile, lyophilized formulations of a compositionthat are suitable for reconstitution. A kit may also contain one or morebuffers suitable for reconstitution and/or dilution of other reagents.Other containers that may be used include, but are not limited to, apouch, tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

Methods of Disease Tissue Detection, Diagnosis and Imaging

In Vivo Diagnosis

Anti-HIV antibodies or fragments are of use for in vivo diagnosis.Methods of diagnostic imaging with labeled Mabs are well-known. Forexample, in the technique of immunoscintigraphy, anti-HIV antibodies arelabeled with a gamma-emitting radioisotope and introduced into apatient. A gamma camera is used to detect the location and distributionof gamma-emitting radioisotopes. See, for example, Srivastava (ed.),RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING AND THERAPY (Plenum Press1988), Chase, “Medical Applications of Radioisotopes,” in REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition, Gennaro et al. (eds.), pp.624-652 (Mack Publishing Co., 1990), and Brown, “Clinical Use ofMonoclonal Antibodies,” in BIOTECHNOLOGY AND PHARMACY 227-49, Pezzuto etal. (eds.) (Chapman & Hall 1993). Also preferred is the use ofpositron-emitting radionuclides (PET isotopes), such as with an energyof 511 keV, such as fluorine-18 (¹⁸F), gallium-68 (⁶⁸Ga), and iodine-124(¹²⁴I). Such imaging can be conducted by direct labeling of the anti-HIVantibody or fragment, or by a pretargeted imaging method, as describedin Goldenberg et al., 2006, J. Clin. Oncol. 24:823-34; see also U.S.Patent Publication Nos. 20050002945, 20040018557, 20030148409 and20050014207, each incorporated herein by reference.

For diagnostic imaging, radioisotopes may be bound to the anti-HIVantibody or fragment either directly or indirectly by using anintermediary functional group. Useful intermediary functional groupsinclude chelators such as ethylenediaminetetraacetic acid anddiethylenetriaminepentaacetic acid. For example, see Shih et al., supra,and U.S. Pat. No. 5,057,313.

The radiation dose delivered to the patient is maintained at as low alevel as possible through the choice of isotope for the best combinationof minimum half-life, minimum retention in the body, and minimumquantity of isotope which will permit detection and accuratemeasurement. Examples of radioisotopes that can be bound to anti-HIVantibody and are appropriate for diagnostic imaging include ^(99m)Tc and¹¹¹In.

The anti-HIV antibody or fragments thereof also can be labeled withparamagnetic ions and a variety of radiological contrast agents forpurposes of in vivo diagnosis. Contrast agents that are particularlyuseful for magnetic resonance imaging comprise gadolinium, manganese,dysprosium, lanthanum, or iron ions. Additional agents include chromium,copper, cobalt, nickel, rhenium, europium, terbium, holmium, orneodymium. Anti-HIV antibody or fragments thereof can also be conjugatedto ultrasound contrast/enhancing agents. For example, one ultrasoundcontrast agent is a liposome that comprises a humanized anti-HIV IgG orfragment thereof. Also preferred, the ultrasound contrast agent is aliposome that is gas filled.

In a preferred embodiment, a bispecific antibody can be conjugated to acontrast agent. For example, the bispecific antibody may comprise morethan one image-enhancing agent for use in ultrasound imaging. In apreferred embodiment, the contrast agent is a liposome. Preferably, theliposome comprises a bivalent DTPA-peptide covalently attached to theoutside surface of the liposome. Still more preferred, the liposome isgas filled.

Imaging Agents and Radioisotopes

In certain embodiments, the claimed peptides or proteins may be attachedto imaging agents of use for imaging and diagnosis of various diseasedorgans, tissues or cell types. Many appropriate imaging agents are knownin the art, as are methods for their attachment to proteins or peptides(see, e.g., U.S. Pat. Nos. 5,021,236 and 4,472,509, both incorporatedherein by reference). Certain attachment methods involve the use of ametal chelate complex employing, for example, an organic chelating agentsuch a DTPA attached to the protein or peptide (U.S. Pat. No.4,472,509). Proteins or peptides also may be reacted with an enzyme inthe presence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents or by reaction with an isothiocyanate.

Non-limiting examples of paramagnetic ions of potential use as imagingagents include chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and erbium (III), with gadolinium beingparticularly preferred. Ions useful in other contexts, such as X-rayimaging, include but are not limited to lanthanum (III), gold (III),lead (II), and especially bismuth (III).

Radioisotopes of potential use as imaging or therapeutic agents includeastatine²¹¹, ¹⁴carbon, ⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt,copper⁶², copper⁶⁴, copper⁶⁷, ¹⁵²Eu, fluorine¹⁸, gallium⁶⁷, gallium⁶⁸,³hydrogen, iodine¹²³, iodine¹²⁴, iodine¹²⁵, iodine¹³¹, indium“, ⁵²iron,⁵⁹iron, ³²phosphorus, ³³phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸, Sc⁴⁷,⁷⁵selenium, silver”, ³⁵sulphur, technicium^(94m) technicium^(99m)yttrium⁸⁶ and yttrium⁹⁰. ¹²⁵I is often being preferred for use incertain embodiments, and technicium^(99m) and indium¹¹¹ are also oftenpreferred due to their low energy and suitability for long rangedetection.

Radioactively labeled proteins or peptides may be produced according towell-known methods in the art. For instance, they can be iodinated bycontact with sodium or potassium iodide and a chemical oxidizing agentsuch as sodium hypochlorite, or an enzymatic oxidizing agent, such aslactoperoxidase. Proteins or peptides may be labeled withtechnetium^(99m) by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the peptide to this column or bydirect labeling techniques, e.g., by incubating pertechnate, a reducingagent such as SNCl₂, a buffer solution such as sodium-potassiumphthalate solution, and the peptide. Intermediary functional groupswhich are often used to bind radioisotopes which exist as metallic ionsto peptides include diethylenetriaminepentaacetic acid (DTPA), DOTA,NOTA, porphyrin chelators and ethylene diaminetetracetic acid (EDTA).Also contemplated for use are fluorescent labels, including rhodamine,fluorescein isothiocyanate and renographin.

In certain embodiments, the anti-HIV antibodies or fragments may belinked to a secondary binding ligand or to an enzyme (an enzyme tag)that will generate a colored product upon contact with a chromogenicsubstrate. Examples of suitable enzymes include urease, alkalinephosphatase, (horseradish) hydrogen peroxidase and glucose oxidase.Preferred secondary binding ligands are biotin and avidin orstreptavidin compounds. The use of such labels is well known to those ofskill in the art in light and is described, for example, in U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149and 4,366,241; each incorporated herein by reference. These fluorescentlabels are preferred for in vitro uses, but may also be of utility in invivo applications, particularly endoscopic or intravascular detectionprocedures.

In alternative embodiments, anti-HIV antibody or fragments may be taggedwith a fluorescent marker. Non-limiting examples of photodetectablelabels include Alexa 350, Alexa 430, AMCA, aminoacridine, BODIPY630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX,5-carboxy-4′,5′-dichloro-2′,7′-dimethoxy fluorescein,5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein,5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethyl amino,Cascade Blue, Cy2, Cy3, Cy5,6-FAM, dansyl chloride, Fluorescein, HEX,6-JOE, NBD (7-nitrobenz-2-oxa-1,3-diazole), Oregon Green 488, OregonGreen 500, Oregon Green 514, Pacific Blue, phthalic acid, terephthalicacid, isophthalic acid, cresyl fast violet, cresyl blue violet,brilliant cresyl blue, para-aminobenzoic acid, erythrosine,phthalocyanines, azomethines, cyanines, xanthines, succinylfluoresceins,rare earth metal cryptates, europium trisbipyridine diamine, a europiumcryptate or chelate, diamine, dicyanins, La Jolla blue dye,allopycocyanin, allococyanin B, phycocyanin C, phycocyanin R, thiamine,phycoerythrocyanin, phycoerythrin R, REG, Rhodamine Green, rhodamineisothiocyanate, Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), Tetramethylrhodamine, and Texas Red. These andother luminescent labels may be obtained from commercial sources such asMolecular Probes (Eugene, Oreg.).

Chemiluminescent labeling compounds of use may include luminol,isoluminol, an aromatic acridinium ester, an imidazole, an acridiniumsalt and an oxalate ester, or a bioluminescent compound such asluciferin, luciferase and aequorin.

In various embodiments, labels of use may comprise metal nanoparticles.Methods of preparing nanoparticles are known. (See e.g., U.S. Pat. Nos.6,054,495; 6,127,120; 6,149,868; Lee and Meisel, J. Phys. Chem.86:3391-3395, 1982.) Nanoparticles may also be obtained from commercialsources (e.g., Nanoprobes Inc., Yaphank, N.Y.; Polysciences, Inc.,Warrington, Pa.). Modified nanoparticles are available commercially,such as NANOGOLD® (gold nanoparticles) from Nanoprobes, Inc. (Yaphank,N.Y.). Functionalized nanoparticles of use for conjugation to proteinsor peptides may be commercially obtained.

Cross-Linkers

In some embodiments, anti-HIV antibodies or fragments or other HIVtargeting molecules may be labeled using various cross-linking reagentsknown in the art, such as homo-bifunctional, hetero-bifunctional and/orphotoactivatable cross-linking reagents. Non-limiting examples of suchreagents include bisimidates; 1,5-difluoro-2,4-(dinitrobenzene);N-hydroxysuccinimide ester of suberic acid; disuccinimidyl tartarate;dimethyl-3,3′-dithio-bispropionimidate;N-succinimidyl-3-(2-pyridyldithio)propionate;4-(bromoaminoethyl)-2-nitrophenylazide; and 4-azidoglyoxal. In anexemplary embodiment, a carbodiimide cross-linker, such as DCCD or EDC,may be used to cross-link acidic residues to amino or other groups. Suchreagents may be modified to attach various types of labels, such asfluorescent labels.

Bifunctional cross-linking reagents have been extensively used for avariety of purposes. Homobifunctional reagents that carry two identicalfunctional groups proved to be highly efficient in inducingcross-linking between identical and different macromolecules or subunitsof a macromolecule, and linking of polypeptide ligands to their specificbinding sites. Heterobifunctional reagents contain two differentfunctional groups. By taking advantage of the differential reactivitiesof the two different functional groups, cross-linking can be controlledboth selectively and sequentially. The bifunctional cross-linkingreagents can be divided according to the specificity of their functionalgroups, e.g., amino, sulfhydryl, guanidino, indole, carboxyl specificgroups. Of these, reagents directed to free amino groups have becomeespecially popular because of their commercial availability, ease ofsynthesis and the mild reaction conditions under which they can beapplied. A majority of heterobifunctional cross-linking reagentscontains a primary amine-reactive group and a thiol-reactive group.

In another example, heterobifunctional cross-linking reagents andmethods of using the cross-linking reagents are described (U.S. Pat. No.5,889,155, incorporated herein by reference). The cross-linking reagentscombine a nucleophilic hydrazide residue with an electrophilic maleimideresidue, allowing coupling in one example, of aldehydes to free thiols.The cross-linking reagent can be modified to cross-link variousfunctional groups.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered to function well in the practice of the invention,and thus can be considered to constitute preferred modes for itspractice. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1 Inhibition of HIV Infection In Vitro and In Vivo UsingConjugated Anti-HIV Antibodies

Summary

To demonstrate the efficacy of an immunoconjugate against HIV-1, murinemonoclonal antibody (Mab) against the envelope antigen of HIV (P4/D10)was conjugated with the conventional anti-cancer drug, doxorubicin, andtested against infectious virus and infected cells, both in vitro and invivo. P4/D10 antibody was incubated with free virus (neutralization) orHIV-infected cells (inhibition) and the resulting infection was measuredby a p24 capture enzyme-linked immunosorbent assay. In an HIV-1/MuLVmouse challenge model the ability of the conjugate to inhibit infectionin vivo was measured.

Doxorubicin-conjugated P4/D10 neutralized HIV-1_(IIIB) and eliminatedintercellular spread and HIV replication in infected Jurkat cells invitro. It also protected mice from challenge with HIV-1_(IIIB)/MuLV atan eight-fold lower concentration than needed for free antibody, whereasno effects were observed for free drug or irrelevant conjugate controls.

These results demonstrate that doxorubicin was concentrated toHIV-infected cells by the P4/D10 antibody, significantly (p=0.0001)contributing to HIV elimination. The same compositions and methods areof use to eradicate remaining antigen-expressing T-cells in patientstreated with ART (anti-retroviral therapy).

In this study, we conjugated doxorubicin, an anticancer anthracyclinewith known pharmacology, toxicology, and antitumor activity in patients,to a neutralizing and ADCC-mediating monoclonal antibody (Mab) developedagainst the HIV-1 outer envelope gp120 (third variable loop region).

The P4/D10 antibody conjugated to doxorubicin was tested in vitro forits efficacy in eliminating HIV-1-infected cells among non-infectedcells and in a mouse model by removing HIV-1/MuLV (murine leukemiavirus) infected syngeneic cells from the intraperitoneal cavity. Theanti-gp120 antibody, P4/D10, neutralizes HIV-1 virus and mediates ADCC(Broliden et al., 1990). It has also been used in its unconjugated formin a phase-I clinical trial for late-stage HIV-1 infected individuals,where it decreased HIV antigens for an extended period of time (Hinkulaet al., 1994). The present study was the first to examine thecombination of P4/D10 in a drug-conjugated form in a preclinical HIVmodel, in comparison to free Mab, free drug, and the irrelevantantibodies hRS7 (Stein et al., Int J Cancer 1993, 55:938-946) and hLL1Griffiths et al., Clin Cancer Res 2003, 9:6567-6571; Sapra et al., ClinCancer Res 2005, 11:5257-5264), that were conjugated similarly withdoxorubicin.

Materials and Methods

Antibodies and Drug Conjugation.

Conjugation of doxorubicin with the IgG1K anti-gp120 antibody P4/D10(Broliden et al., 1990) and the control antibodies, as well as thepreparation of the bifunctional doxorubicin hydrazone derivative with amaleimide group, were performed according to Griffiths et al. (2003).Briefly, antibodies P4/D10, hLL1 (humanized anti-CD74), and hRS7(humanized anti-EGP-1) in a final concentration of approximately 9mg/ml, were mildly reduced with DTT (dithiothreitol) in PBS (pH 7.5)containing 5 mM EDTA, using about 2.2 mM final DTT concentration,corresponding to a 38-fold molar excess of the reductant with respect tothe antibodies. The solutions were incubated at 37° C. for 40 min. Thereduced Mabs were purified on spin-columns of Sephadex G50/80 in 50 mMsodium acetate buffer containing 150 mM NaCl and 2 mM EDTA (pH 5.3). Thenumber of thiol groups generated on the antibodies was determined byEllman's assay. For conjugation, mildly reduced antibodies at 6.5 mg/mlwere mixed with the bifunctional doxorubicin. The incubates were kept onice for 15 min, and purified on spin columns of G50/80 in 0.1 M sodiumacetate (pH 6.5), followed by passage through a short column ofBio-Beads SM2 (Bio-Rad, Hercules, Calif.) equilibrated in the samebuffer. The products were analyzed for doxorubicin/Mab substitutionratios by measuring absorbance. Size-exclusion HPLC analyses wereperformed on an analytical BioSil 250 column.

A GMP-produced lot of IgG from HIV infected patients (HIVIgG) (Guay etal. AIDS 2002, 16:1391-1400) was used as positive control and sera fromHIV-negative individuals as negative controls. Free doxorubicin, as wellas the anticancer humanized Mabs LL1 and RS7, similarly conjugated withdoxorubicin, were included as controls for the conjugated P4/D10antibody.

HIV-1 Neutralization Assay.

Doxorubicin P4/D10, unlabelled P4/D10, HIV immunoglobulin (HIVIgG), andHIV-negative serum were mixed with the HIV-1 isolate HIV-1_(IIIB) (LAI)and incubated for 1 h at 37° C. before 50,000 Jurkat T-cells/well wereadded. After 1 h of incubation, the cells were washed with medium andnew complete medium added (200 μl/well). After 7 days of culture, theamount of p24 produced was measured by a p24 capture ELISA(enzyme-linked immunosorbent assay) and the percent inhibition of HIV-1p24 production was calculated.

HIV-1 Inhibition In Vitro.

Jurkat T-cells were infected with HIV-1_(IIIB) by mixing 5-10×10⁶ cellswith 100× TCID₅₀ HIV-1_(IIIB) and incubating for 1 h at 37° C. The cellswere washed in medium and incubated at 37° C. Every third day, mediumwas changed and supernatant checked for p24 production. When close to100% of the cells were infected, different proportions ofHIV-1_(IIIB)-infected cells were mixed with uninfected cells. The cellswere treated with serial dilutions of antibodies, serum, or freedoxorubicin from 100 to 0.00001 μg/ml. After seven days of culture at37° C., HIV-1 p24 inhibition was measured and supernatants from cellspreviously treated with 0.1-10 μg/ml of doxorubicin-P4/D10, unconjugatedP4/D10, and 0.05-0.5 mg/ml HIV-negative serum were collected andtransferred to fresh Jurkat T-cells to test if infectious HIV wasidentified by the p24 ELISA at days 3, 7, 10, 12, and 15 afterinitiation of the culture.

HIV-1/MuLV Challenge Model.

A human T-cell line, CEM-1B, with a genetically integrated MuLV genomewas infected with HIV-1_(IIIB), which led to the production ofpseudoviruses with the HIV-1 genome and the MuLV envelope (Adang et al.,PNAS USA 1999, 96:12749-753; Hinkula et al., Cells Tissues Organs 2004,177:169-184). These virus supernatants were used to infect splenocytesfrom C57Bl/6×DBA F1 K^(b/d) mice transgenic for HLA-A201. Isogenic micewere challenged with HIV-1_(IIIB)/MuLV infected splenocytes i.p. andwere immediately given conjugated antibodies, free antibodies or freedoxorubicin i.p. Ten days after challenge, mice were sacrificed andperitoneal cells collected. Peritoneal cells were pelleted and added to1×10⁶ HIV susceptible Jurkat T-cells or human PBMC grown in 24-wellplates. From these secondary cultures, supernatant was removed and freshmedium added every 3-4 days. The amount of infectious HIV recovered inthe supernatant was measured for 3 weeks by p24 ELISA.

Statistical Analysis.

To compare the in vitro HIV-1 neutralizing capacities of the anti-gp120Mabs and control antibodies, Student's t-test and the non-parametricKruskal-Wallis test were used. Statistical comparisons between thegroups of mice treated with different antibodies were performed usingthe nonparametric Mann-Whitney U and Kruskal-Wallis tests. A differencewas considered significant when a p-value of <0.05 was obtained. Anon-parametric one-way ANOVA test was performed using GraphPad Prismversion 4.0a (GraphPad Software, San Diego, Calif.) and was used forcomparisons of HIV-1 isolation and p24 antigen positivity between thestudy groups.

Results

The number of thiol groups generated on the respective antibodies bymild reduction, as well as the doxorubicin/Mab substitution ratios inthe final purified conjugates, ranged between 8.8 (P4/D10, hRS7) and 9.4(hLL1), giving a ratio of approximately 9 drug molecules per IgG.High-pressure liquid chromatographic analyses showed that the conjugatesand the native Mabs possessed similar retention times, with zero tominimal aggregation (data not shown).

No significant difference in HIV-1 neutralizing capacity of free HIV-1virus (FIG. 1A) could be shown between the doxorubicin-conjugated P4/D10Mab and either unconjugated P4/D10 Mab or the HIVIgG antibodies.However, all anti HIV-1 specific antibodies were significantly betterthen the negative control serum (p=0.001) at neutralizing HIV-1_(IIIB).

When 3% HIV-1_(IIIB) infected Jurkat cells were mixed with 97%uninfected cells, doxorubicin-P4/D10 mediated a significantly (p=0.002)stronger inhibition of intercellular spread of HIV-1 infection than freeP4/D10, doxorubicin-conjugated control antibody, hLL1, or freedoxorubicin at a concentration of 0.5 or 0.05 μg/ml (FIG. 1B). Similarresults were seen at all other concentrations of infected and uninfectedcells. It was of particular interest that the intercellular spread ofinfection appeared to be inhibited even more potently than the effectobtained with doxorubicin-P4/D10 as a neutralizing agent. Also, noinfectious virus could be found in the cultures treated with high dosesof doxorubicin-P4/D10, since no p24 production was detected aftertransfer of supernatants from these cell cultures to uninfected Jurkatcells (data not shown). The significant difference in effect betweendoxorubicin-P4/D10 and unconjugated P4/D10 could not have been predictedfrom the results on neutralization of free HIV-1 virus (FIG. 1A).

To test the efficacy of doxorubicin-P4/D10 antibody in vivo, mice weregiven isogeneic HIV/MuLV-infected cells together with conjugatesintraperitoneally. Peritoneal cells were harvested 10 days later andinfectious HIV was demonstrated in all controls, similar to previousstudies (Hinkula et al., 2004). The doxorubicin-P4/D10 antibodyprotected mice completely against challenge with HIV-1 infected primarylympoid cells (p=0.0001) (FIG. 2). No infectious HIV was recovered fromperitoneal cells after challenge and treatment with 100 μg ofdoxorubicin-P4/D10 antibody. When mice were treated with 100 μg ofunconjugated P4/D10 antibody, all were positive for p24 production.Complete protection by antibody alone was seen only when the dose wasincreased eight-fold, to 800 μg unconjugated P4/D10 per mouse. None ofthe doxorubicin-conjugated control antibodies (hLL1 or hRS7) providedany protection at doses of 100-200 μg, nor did doses of 100-400 μg offree doxorubicin.

Discussion

The results above show that the total virus-inhibiting properties of anantibody directed against the envelope of HIV-1 could be amplifiedsignificantly by coupling to doxorubicin. Doxorubicin-P4/D10 was capableof eliminating HIV-1 infected cells in vitro, as well as in anexperimental in vivo challenge model. The ability of the unconjugatedP4/D10 Mab to mediate ADCC against HIV-1 infected target cells as wellas neutralizing HIV-1 (Broliden et al., 1990; Hinkula et al., 1994) mayenhance its efficacy as a drug immunoconjugate in a non-toxic manner.

An anticancer anti-CD74 Mab, LL1, conjugated similarly to doxorubicin,has at very low doses shown remarkable activity in vitro and in humanxenograft models of non-Hodgkin's lymphoma or multiple myeloma(Griffiths et al., 2003; Sapra et al., 2005). These studies, as well astoxicology in monkeys, indicated that only very high doses of theimmunoconjugate would result in evidence of bone marrow suppression, butno cardiac toxicity related to doxorubicin was observed (Sapra et al.,2005). To avoid virus escape, antibodies directed against both conservedand variable sites of accessible epitopes of HIV should be testedtogether (Trkola et al., 2005; Ferrantelli et al., J Infect Dis 2004,189:2167-2173).

In previous in vitro studies, HIV-1 specific immunoglobulins conjugatedto Pseudomonas exotoxin A (PE40) removed HIV-1 infected cells (Pincus etal., 2003; Ashorn et al., Proc Natl Acad Sci USA 1990, 87:8889-8893).However, in clinical trials, PE40 coupled to CD4 cells proved to beimmunogenic and hepatotoxic (Davey et al., 1994; Ramachandran et al.,1994). Thus, the present results, showing efficacy of a doxorubicin-Mabconjugate with little or no side effects, are surprising and unexpected.The toxicity of PE40-CD4 might be explained by formation of toxiccomplexes with free gp120, which is present in high concentrations innon-ART treated patients with high viral load (Berger et al., 1998). Inorder to avoid potential toxicity problems associated with high viralloads, molecules targeting HIV-envelope-specific epitopes shouldpreferably be used in the setting when viral burden is low, such asduring ART, early in HIV infection or even shortly after a known orpotential exposure to infection with HIV. For example, health careworkers exposed to accidental needle stick with HIV-contaminated orpotentially contaminated blood or other fluids may be treated with aconjugated antibody according to the disclosed methods. As a result ofthe anticellular activity of the doxorubicin-P4/D10 conjugate, theaddition of a drug conjugate to patients treated with ART may eliminateantigen-carrying cells as well as free virions, and thus reduce theviral load even further. The skilled artisan will realize that theclaimed compositions and methods are not limited to a doxorubicinconjugate of P4/D10, but rather may utilize other known cytotoxic agentsconjugated to P4/D10 or to other known anti-HIV antibodies.

In other embodiments, in order to avoid non-specific toxicity,bispecific antibodies and other pretargeting strategies may be used, inwhich antibody targeting and delivery of the toxic agent are separated(Wu et al., 2005). This strategy has shown promising results both inpreclinical and in clinical cancer trials (Forero et al., Blood 2004,104:227-236; Rossi et al., Clin Cancer Res 2005, 11:7122s-7129s). For invivo use in human subjects, human or humanized forms of antibody forrepeated clinical use are preferred.

Example 2 Treatment of an HIV-Infected Patient Following ART

A 47-year old male patient is determined to be seropositive for HIV. Thepatient has a CD4 count of less than 200/mm³. The patient is treatedwith a standard regimen of the non-nucleoside reverse transcriptaseinhibitor nevirapine. CD4 cell count improves to 300/mm³, but thepatient is still seropositive for HIV. The patient is treated withhumanized doxorubicin-P4/D10 antibody. The patient's CD4 count improvesto over 350/mm³ and the patient is no longer seropositive for HIV. Oneyear later, the patient remains asymptomatic and there is no detectablepresence of HIV infection.

Example 3 Treatment of a Health Care Worker after Accidental NeedleStick

A 30-year old nurse practitioner is exposed to an accidental needlestick with HIV positive blood. Within 1 hour, the subject is treatedwith humanized doxorubicin-P4/D10 antibody. One year later, there is nosign of HIV infection in the subject.

Example 4 Treatment with Other Cytotoxins and/or Antibodies

A 28-year old male patient is determined to be seropositive for HIV. Thepatient has a CD4 count of less than 200/mm³. The patient is treatedwith a standard regimen of the non-nucleoside reverse transcriptaseinhibitor nevirapine. CD4 cell count improves to 300/mm³, but thepatient is still seropositive for HIV. The patient is treated with abispecific antibody comprising a humanized 4E10 Fab-734 scFv, preparedusing methods disclosed in U.S. Pat. No. 7,052,872 (incorporated hereinby reference). A 24 hour incubation after injection was used to allowfree bispecific antibody to clear from circulation, followed byinjection of 5-fluorouracil conjugated to targeting peptide IMP-156(Id.). The patient's CD4 count improves to over 350/mm³ and the patientis no longer seropositive for HIV. One year later, the patient remainsasymptomatic and there is no detectable presence of HIV infection.

Example 5 Pretargeting with IMP411

A 35-year old male patient is determined to be seropositive for HIV. Thepatient has a CD4 count of less than 180/mm³. The patient is treatedwith a standard regimen of the non-nucleoside reverse transcriptaseinhibitor nevirapine. CD4 cell count improves to 250/mm³, but thepatient is still seropositive for HIV. The patient is treated with abispecific antibody comprising a bispecific anti-gp120 P4/D10IgG1×Fab-679, prepared by the DNL technique using methods disclosed inU.S. patent application Ser. Nos. 11/389,358, 11/391,584, 11/478,021 and11/633,729, incorporated herein by reference. A 24 hour incubation afterinjection was used to allow free bispecific antibody to clear fromcirculation, followed by injection of SN38 conjugated to targetingpeptide IMP-411. The patient's CD4 count improves to over 325/mm³ andthe patient is no longer seropositive for HIV. One year later, thepatient remains asymptomatic and there is no detectable presence of HIVinfection.

Example 6 Humanization of Anti-HIV Antibody

cDNAs encoding the VL and VH regions of a mouse monoclonal antibodyagainst HIV envelope protein are isolated and separately recombinantlysubcloned into mammalian expression vectors containing the genesencoding kappa and IgG₁ constant regions, respectively, of humanantibodies. Cotransfection of mammalian cells with these two recombinantDNAs results in expression of a humanized Mab that has the same bindingand therapeutic characteristics of the parent mouse Mab.

The CDRs of the VK and VH DNAs are recombinantly linked to the framework(PR) sequences of the human VK and VH regions, respectively, which aresubsequently linked, respectively, to the human kappa and IgG₁ constantregions, so as to express in mammalian cells. Generally, as discussedherein, “chimeric” Mabs are formed by joining or subcloning murine VKand VH regions to human constant light and heavy chains, respectively,while “humanized” Mabs are further derivatized by replacing the murineframework (FR) sequences in the chimeric Mab with the correspondinghuman FR sequences. As discussed below, the humanized Mab may be furtheroptimized by replacement of one or more human FR amino acids withcorresponding murine FR amino acids, particularly for FR residuestouching or close to the CDR amino acid residues.

In various embodiments, antibody variable domains can be modeled bycomputer modeling (see, for example, Dion, “Humanization of MonoclonalAntibodies: Molecular Approaches and Applications,” in Goldenberg et al.eds., Cancer Therapy With Radiolabeled Antibodies, Ch. 19, CRC Press,Boca Raton, Fla., 1994), which is incorporated herein by reference. Ingeneral, the 3-D structure for Mabs are best modeled by homology,preferably following identification of human FR sequences showing high(over 75%, preferably over 85%, more preferably over 90%, morepreferably over 95%) homology with the murine FR sequences to bereplaced. Wherever possible, side group replacements should be performedso as to maintain the torsion angle between Cα and Cβ. Energyminimization may be accomplished by the AMBER forcefield (Weiner et al,J. Amer. Chem. Soc. 106: 765, 1984) using the convergent method.Potentially critical FR-CDR interactions can be determined by initiallymodeling the light and heavy variable chains. All murine FR residueswithin a 4.5 angstrom radius of all atoms within each CDR can thereby beidentified and retained in the final design model of the humanizedantibody.

Once the sequences for the VK and VH domains are designed, CDRengrafting can be accomplished by gene synthesis using long syntheticDNA oligonucleotides as templates and short oligonucleotides as primersin a PCR reaction. In most cases, the DNA encoding the VK or VH domainwill be approximately 350 bp long. By taking advantage of codondegeneracy, a unique restriction site may easily be introduced, withoutchanging the encoded amino acids, at regions close to the middle of theV gene DNA sequence. Two long non-overlapping single-stranded DNAoligonucleotides about 150 bp upstream and downstream of the restrictionsite can be generated by automated DNA oligonucleotide synthesizer(Cyclone Plus DNA Synthesizer, Milligen-Biosearch). As the yields offull length DNA oligonucleotides may be expected to be low, they can beamplified by two pairs of flanking primers in a PCR reaction.

The primers can be designed with the necessary restriction sites tofacilitate subsequent subcloning. Primers for oligo A and for oligo Bshould contain overlapping sequence at the restriction site so that theresultant PCR product for oligo A and B, respectively, can be joinedin-frame at the restriction site to form a full length DNA sequence (ca350 bp) encoding the VH domain.

The ligation of the PCR products for oligo A and B and their subcloninginto appropriate restriction sites of the staging vector can becompleted in a single three-fragment-ligation step. The subcloning ofthe correct sequence into the staging vector can be first analyzed byrestriction digestion analysis and subsequently confirmed by sequencingreaction according to Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463(1977).

A restriction fragment containing the Ig promoter, leader sequence andthe VH sequence can be excised from the staging vector and subcloned tothe corresponding sites in a pSVgpt-based vector, which contains thegenomic sequence of the human IgG constant region, an Ig enhancer and agpt selection marker, forming the final expression vector. Similarstrategies can be employed for the construction of the VK sequence.

The DNA sequence containing the Ig promoter, leader sequence and theanti-HIV VK sequence can be excised from the staging vector by treatmentwith appropriate endonucleases, and can be subcloned into thecorresponding sites of a pSVhyg-based vector, pKh, which contains thegenomic sequence of human kappa chain constant regions, a hygromycinselection marker, an Ig and a kappa enhancer, forming the finalexpression vector.

As humanization sometimes results in a reduction or even loss ofantibody affinity, additional modification might be required in order torestore the original affinity (See, for example, Tempest et al.,Bio/Technology 9: 266 (1991); Verhoeyen et al., Science 239: 1534(1988)), which are incorporated by reference. In general, to preparechimeric anti-HIV Mab, VH and VK chains of the anti-HIV Mab can beobtained by PCR cloning using DNA products and primers. Orlandi et al.(Proc. Natl. Acad. Sci., USA, 1989, 86: 3833), and Leung et al.(BioTechniques, 1993, 15:286). The VK PCR primers may be subcloned intoa pBR327 based staging vector (VKpBR) as described above. The VH PCRproducts may be subcloned into a similar pBluescript-based stagingvector (VHpBS) as described above. The fragments containing the VK andVH sequences, along with the promoter and signal peptide sequences, canbe excised from the staging vectors using appropriate restrictionendonucleases. The VK fragments (about 600 bp) can be subcloned into amammalian expression vector (for example, pKh) conventionally. pKh is apSVhyg-based expression vector containing the genomic sequence of thehuman kappa constant region. an Ig enhancer, a kappa enhancer and thehygromycin-resistant gene. Similarly, the about 800 bp VH fragments canbe subcloned into pG1g, a pSVgpt-based expression vector carrying thegenomic sequence of the human IgG1 constant region, an Ig enhancer andthe xanthine-guanine phosphoribosyl transferase (gpt) gene. The twoplasmids may be transfected into mammalian expression cells, such asSp2/O—Ag14 cells, by electroporation and selected for hygromycinresistance. Colonies surviving selection are expanded, and supernatantfluids monitored for production of chimeric anti-HIV Mab by an ELISAmethod. A transfection efficiency of about 1-10×10⁶ cells is desirable.An antibody expression level of between 0.10 and 2.5 μg/ml can beexpected with this system.

RNA isolation, cDNA synthesis, and amplification can be carried out asfollows. Total cell RNA can be prepared from a anti-HIV hybridoma cellline, using a total of about 10⁷ cells, according to Sambrook et al.,(Molecular Cloning: A Laboratory Manual, Second ed., Cold Spring HarborPress, 1989), which is incorporated by reference. First strand cDNA canbe reverse transcribed from total RNA conventionally, such as by usingthe SuperScript preamplification system (Gibco/BRL., Gaithersburg, Md.).Briefly, in a reaction volume of 20 μl, 50 ng of random primers can beannealed to 5 μg of RNAs in the presence of 2 μl of 10× synthesis buffer[200 mM Tris-HCl (pH 8.4), 500 mM KCl, 25 mM MgCl₂, 1 mg/ml BSA], 1 μlof 10 mM dNTP mix, 2 μl of 0.1 M DTT, and 200 units of SuperScriptreverse transcriptase. The elongation step is initially allowed toproceed at room temperature for 10 mM followed by incubation at 42° C.for 50 mM. The reaction can be terminated by heating the reactionmixture at 90° C. for 5 mM.

The VK and VH sequences for chimeric or human anti-HIV Mab can amplifiedby PCR as described by Orlandi et al., (Proc. Natl. Acad. Sci., USA, 86:3833 (1989)) which is incorporated by reference. VK sequences may beamplified using the primers CK3BH and VK5-3 (Leung et al.,BioTechniques, 15: 286 (1993), which is incorporated by reference),while VH sequences can be amplified using the primer CH1B which annealsto the CH1 region of murine IgG, and VHIBACK (Orlandi et al., 1989). ThePCR reaction mixtures containing 10 μl of the first strand cDNA product,9 μl of 10× PCR buffer [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mMMgCl2, and 0.01% (w/v) gelatin] (Perkin Elmer Cetus, Norwalk, Conn.),can be subjected to 30 cycles of PCR. Each PCR cycle preferably consistsof denaturation at 94° C. for 1 min, annealing at 50° C. for 1.5 min,and polymerization at 72° C. for 1.5 min. Amplified VK and VH fragmentscan be purified on 2% agarose (BioRad, Richmond, Calif.).

PCR products for VK can be subcloned into a staging vector, such as apBR327-based staging vector VKpBR that contains an Ig promoter, a signalpeptide sequence and convenient restriction sites to facilitate in-frameligation of the VK PCR products. PCR products for VH can be subclonedinto a similar staging vector, such as the pBluescript-based VHpBS.Individual clones containing the respective PCR products may besequenced by, for example, the method of Sanger et al., Proc. Natl.Acad. Sci., USA, 74: 5463 (1977) which is incorporated by reference.

The two plasmids can be co-transfected into an appropriate cell, e.g.,myeloma Sp2/O—Ag14, colonies selected for hygromycin resistance, andsupernatant fluids monitored for production of chimeric or humanizedanti-HIV antibodies by, for example, an ELISA assay.

Transfection and assay for antibody secreting clones by ELISA, can becarried out as follows. About 10 μg of light chain expression vector and20 μg of heavy chain expression vector can be used for the transfectionof 5×10⁶ SP2/O myeloma cells by electroporation (BioRad, Richmond,Calif.) according to Co et al., J. Immunol., 148: 1149 (1992) which isincorporated by reference. Following transfection, cells may be grown in96-well microtiter plates in complete HSFM medium (GIBCO, Gaithersburg,Md.) at 37° C., 5% CO₂. The selection process can be initiated after twodays by the addition of hygromycin selection medium (Calbiochem, SanDiego, Calif.) at a final concentration of 500 μg/ml of hygromycin.Colonies typically emerge 2-3 weeks post-electroporation. The culturescan then be expanded for further analysis.

Transfectoma clones that are positive for the secretion of chimeric orhumanized heavy chain can be identified by ELISA assay. Briefly,supernatant samples (100 μl) from transfectoma cultures are added intriplicate to ELISA microtiter plates precoated with goat anti-human(GAH)-IgG, F(ab′)₂ fragment-specific antibody (Jackson ImmunoResearch,West Grove, Pa.). Plates are incubated for 1 h at room temperature.Unbound proteins are removed by washing three times with wash buffer(PBS containing 0.05% polysorbate 20). Horseradish peroxidase (HRP)conjugated GAH-IgG, Fc fragment-specific antibodies (JacksonImmunoResearch, West Grove, Pa.) are added to the wells, (100 μl ofantibody stock diluted×10⁴, supplemented with the unconjugated antibodyto a final concentration of 1.0 μg/ml). Following an incubation of 1 h,the plates are washed, typically three times. A reaction solution, [100μl, containing 167 μg of orthophenylene-diamine (OPD) (Sigma, St. Louis,Mo.), 0.025% hydrogen peroxide in PBS], is added to the wells. Color isallowed to develop in the dark for 30 minutes. The reaction is stoppedby the addition of 50 μl of 4 N HCl solution into each well beforemeasuring absorbance at 490 nm in an automated ELISA reader (Bio-Tekinstruments, Winooski, Vt.). Bound chimeric antibodies are thandetermined relative to an irrelevant chimeric antibody standard(obtainable from Scotgen, Ltd., Edinburg, Scotland).

Antibodies can be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof chimeric antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2 micron membrane. The filtered medium is passedthrough a protein A column (1×3 cm) at a flow rate of 1 ml/min. Theresin is then washed with about 10 column volumes of PBS and proteinA-bound antibody is eluted from the column with 0.1 M glycine buffer (pH3.5) containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μl of 3 M Tris(pH 8.6), and protein concentrationsdetermined from the absorbancies at 280/260 nm. Peak fractions arepooled, dialyzed against PBS, and the antibody concentrated, forexample, with the Centricon 30 (Amicon, Beverly, Mass.). The antibodyconcentration is determined by ELISA, as before, and its concentrationadjusted to about 1 mg/ml using PBS. Sodium azide, 0.01% (w/v), isconveniently added to the sample as preservative.

Comparative binding affinities of the mouse, chimeric and humanizedantibodies thus isolated may be determined by direct radioimmunoassay.The chimeric and humanized anti-HIV antibodies are determined to havethe same binding specificity and affinity as the mouse Mab.

All of the COMPOSITIONS and METHODS disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods have been described interms of preferred embodiments, it is apparent to those of skill in theart that variations maybe applied to the COMPOSITIONS and METHODS and inthe steps or in the sequence of steps of the methods described hereinwithout departing from the concept, spirit and scope of the invention.More specifically, certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. A pharmaceutical composition comprising a P4/D10antibody or fragment thereof against an HIV surface envelope antigen,the antibody or fragment conjugated to a cytotoxic drug, whereinexposure to the conjugated antibody or fragment is effective to reduceHIV infection or to limit the intercellular transmission of HIV touninfected cells when administered to a subject.
 2. The composition ofclaim 1, wherein the subject is a human subject.
 3. The composition ofclaim 1, wherein the conjugated antibody or fragment is a chimeric,humanized or human antibody or fragment.
 4. The composition of claim 1,wherein the cytotoxic drug is doxorubicin.
 5. The composition of claim1, wherein the cytotoxic drug is aplidin, azaribine, anastrozole,azacytidine, bleomycin, bortezomib, bryostatin-1, busulfan,calicheamycin, camptothecin, 10-hydroxycamptothecin, carmustine,celebrex, chlorambucil, cisplatin, irinotecan (CPT-11), SN-38,carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine,docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin,doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide,estramustine, etoposide, etoposide glucuronide, etoposide phosphate,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, fluorouracil, gemcitabine, hydroxyurea, idarubicin,Ifosfamide, L-asparaginase, leucovorin, lomustine, mechlorethamine,melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,mithramycin, mitomycin, mitotane, phenyl butyrate, procarbazine,paclitaxel, pentostatin, PSI-341, semustine, streptozocin, tamoxifen,taxanes, taxol, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, velcade, vinblastine, vinorelbine,vincristine or a combination thereof.
 6. The composition of claim 1,wherein the antibody or fragment is a bispecific antibody, a bispecificantibody fragment, an scFv, a Fab, a Fab′, a F(ab)₂, a F(ab′)₂, an Fv,an sFv, an scFv, an scFv-Fc fusion, a single chain antibody, a diabody,a triabody or a tetrabody.
 7. The composition of claim 1, wherein theantibody or fragment is a neutralizing antibody.
 8. The composition ofclaim 1, further comprising a second antibody or fragment thereof,wherein the second antibody or fragment binds to an HIV surface envelopeantigen.
 9. The composition of claim 8, wherein the second antibody orfragment binds to the same HIV surface envelope antigen as the P4/D10antibody.
 10. The composition of claim 8, wherein the second antibody orfragment binds to a different HIV surface envelope antigen than theP4/D10 antibody.
 11. The composition of claim 8, wherein the secondantibody or fragment thereof is selected from the group consisting of4E10, 2F5, 3D6, C37, 1ACY, 1F58, 1GGGC, 2G12 and X5.
 12. The compositionof claim 1, further comprising an anti-retroviral agent selected fromthe group consisting of efavirenz, zidovudine, tenofovir, lamivudine,emtricitabine, didanosine, abacavir, stavudine, nevirapine, lopinavir,ritonavir, atazanavir, fosamprenavir, indinavir, nelfinavir andsaquinavir.
 13. The composition of claim 1, wherein exposure to theconjugated antibody or fragment is effective to reduce HIV infection orto limit the intercellular transmission of HIV to uninfected cells invivo.
 14. The composition of claim 1, wherein the conjugated antibody orfragment thereof is a bispecific antibody or fragment thereof.
 15. Thecomposition of claim 14, wherein the bispecific antibody furthercomprises a second antibody or fragment thereof and the second antibodyor fragment thereof binds to a hapten on a targetable construct.
 16. Thecomposition of claim 14, wherein the bispecific antibody or fragmentcomprises at least one scFv, Fab, Fab′, F(ab)₂, F(ab′)₂, Fv, sFv,scFv-Fc fusion, single chain antibody, diabody, triabody or tetrabody.17. The composition of claim 15, wherein the hapten is HSG(histamine-succinyl-glycine) or In-DTPA.
 18. The composition of claim15, wherein the bispecific antibody or fragment comprises one or more679 antibodies or antibody fragments that bind to HSG.
 19. Apharmaceutical composition comprising an antibody or fragment thereofagainst an HIV surface envelope antigen, the antibody or fragmentconjugated to a cytotoxic drug, wherein the antibody or fragmentcompetes for binding to gp120 with the P4/D10 antibody, wherein exposureto the conjugated antibody or fragment is effective to reduce HIVinfection or to limit the intercellular transmission of HIV touninfected cells when administered to a subject.
 20. The composition ofclaim 19, wherein the subject is a human subject.
 21. The composition ofclaim 19, wherein the conjugated antibody or fragment is a chimeric,humanized or human antibody or fragment.
 22. The composition of claim19, wherein the cytotoxic drug is doxorubicin.
 23. The composition ofclaim 19, wherein the cytotoxic drug is aplidin, azaribine, anastrozole,azacytidine, bleomycin, bortezomib, bryostatin-1, busulfan,calicheamycin, camptothecin, 10-hydroxycamptothecin, carmustine,celebrex, chlorambucil, cisplatin, irinotecan (CPT-11), SN-38,carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine,docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin,doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide,estramustine, etoposide, etoposide glucuronide, etoposide phosphate,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, fluorouracil, gemcitabine, hydroxyurea, idarubicin,ifosfamide, L-asparaginase, leucovorin, lomustine, mechlorethamine,melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,mithramycin, mitomycin, mitotane, phenyl butyrate, procarbazine,paclitaxel, pentostatin, PSI-341, semustine, streptozocin, tamoxifen,taxanes, taxol, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, velcade, vinblastine, vinorelbine,vincristine or a combination thereof.
 24. The composition of claim 19,wherein the antibody or fragment is a bispecific antibody, a bispecificantibody fragment, an scFv, a Fab, a Fab′, a F(ab)₂, a F(ab′)₂, an Fv,an sFv, an scFv, an scFv-Fc fusion, a single chain antibody, a diabody,a triabody or a tetrabody.
 25. The composition of claim 19, wherein theantibody or fragment is a neutralizing antibody.